In the development of three-dimensional cell-polymer synthetic matrices for tissue regeneration, visualization of cells growing in these porous structures can be difficult. The focus of this study was the development and use of a novel method that would allow for visualization of osteoblasts inside opaque matrices. The morphologic responses and phenotypic characterization of osteoblasts as they attach, spread, and migrate through a porous three-dimensional biodegradable polymer-ceramic matrix in vitro were studied using immunofluorescence and confocal laser scanning microscopy (CLSM). CLSM offers several advantages over the most commonly used imaging methods [traditional light microscopy and scanning electron microscopy (SEM)]. CLSM filters out-of-focus background and provides more structural details of cells. In addition, CLSM does not require extensive sample preparation as does SEM. When used in conjunction with fluorescence-labeled antibodies to identify cells and their products, it can characterize morphology of growing cells and successfully determine phenotypic function. Using monoclonal antibody to osteocalcin, a bone cell-specific protein, cells throughout the matrix were found to have preserved osteoblast-like phenotype with growth. The morphology of cells throughout the matrix was found to be similar to osteoblast cells grown on tissue culture polystyrene and consisted of spread polygonal forms. Using the technique of CLSM with immunofluorescent antibodies, we have demonstrated for the first time that these three-dimensional degradable polymer matrices can support osteoblast growth and phenotypic expression throughout its structure. © 1995 John Wiley & Sons, Inc.