Conventional phenotypic screening methods depend largely on visual observations of qualitative traits (Nakazawa et al., 2003). However, subtle trait changes may not be detected by this method. Therefore, mutant candidate lines that require difficult objective evaluation have not been studied. In order to apply a systematic approach to phenotypic analyses, we have proposed a novel method for in silico phenotypic analysis based on 3D shape measurement. This approach includes a 3D laser surface scanner measurement system (Kaminuma et al., 2004), in silico quantitative trait determination via a reconstructed 3D shape model (Kaminuma et al., 2005a), and statistical in silico mutant screening methods (Kaminuma et al., 2005b). In this report, we propose a method for in silico quantitative trait determination using a microfocus computed tomography system (μCT), which functions as a 3D shape-measuring device. Several conventional studies have obtained 2D images of plant inner structures using X-rays (e.g. Koroleva et al., 2000). Allen et al. (2006) used 2D plant root images for practical phenotypic screening. As for 3D based conventional studies, Heeraman et al. (1997) quantified rooting spatial distribution and root length with X-ray CT data in bush bean (Phaseolus vulgaris L.). Similar approaches have been used in chestnut trees (Aesculus hippocastanum L.) and maple (Acer pseudoplatanus L.) (Pierret et al., 1999, 2000). In addition, Stuppy et al. (2003) conducted a survey of high-resolution X-ray CT in plant samples. They reconstructed 3D image data based on X-ray CT; however, in silico phenotypic analysis was not performed. In other words, complete use of 3D image data to reconstruct a 3D shape model or in silico extraction of phenotype was not implemented. In this study, we used μCT-based processing for in silico phenotypic analysis of Arabidopsis thaliana. In order to demonstrate the utility of analytical in silico phenotypic analysis for μCT, 3D-specific leaf trichome traits were proposed. Trichomes are unicellular or multicellular appendages that originate from cells of the aerial epidermis. Population-level studies in plants have indicated that resistance to herbivorous insects is positively correlated with mean trichome density (Valverde et al., 2001). In Arabidopsis, trichome cells are unicellular structures with elusive functions. However, Gutierrez-Alcala et al. (2000) found evidence that biosynthesis occurs in these epidermal structures. Despite the importance of trichome density, most studies of trichome traits are limited to qualitative discussion due to the difficulty of quantification. A successful methodology to quantify trichome traits will greatly facilitate the identification of genes that are integral in trichome control. In this study, two transgenic Arabidopsis lines were evaluated with respect to trichome position, number and size. The CAPRICE (CPC) mutant (Schellmann et al., 2002) is known to possess a large number of trichomes compared with the corresponding wild-type. On the other hand, the GLABRA3 (GL3) mutant displays a reduction in trichome number (Payne et al., 2000). The wild-type, CPC and GL3 mutants were chosen to illustrate how 3D trichome traits can be investigated using both correlation analysis and hypothesis testing for two types of spatial heterogeneity, including the margin, non-margin and mid-rib. In addition, we describe the advantages and disadvantages of μCT as a means for 3D resolution of leaf trichome position. In particular, several important points regarding spatial resolution, data acquisition artefacts and measurement precision are highlighted. Trichome analyses based on μCT can therefore provide new information with regard to trichome phenotypes.