Tooth enamel thickness has long been an important character in studies of primate and especially hominin phylogeny, taxonomy, and adaptation. Current methods for accurately assessing enamel thickness involve the physical sectioning of teeth, because measurements of enamel thickness using some radiographic techniques are unreliable. However, because destructive methods limit sample sizes and access to important fossil specimens, it is desirable that they be replaced with nondestructive techniques. Although microfocal X-ray computed tomography (mCT) has been used recently in studies of enamel thickness, the accuracy of this technique has yet to be established. The present research compares physical sections to computer-generated mCT sections of teeth from a variety of primate and nonprimate, recent and fossil taxa to examine whether enamel thickness, tooth size, and diagenetic remineralization (fossilization) impact the ability of mCT to measure enamel thickness accurately. Results indicate that recent teeth of varying size and thickness are clearly and accurately depicted in mCT scans, with measurements from nearly identical planes in physical and mCT sections differing by 3–5%. A fossil papionin molar (ca. 2 Myr) was also accurately measured using mCT scans, although thinner enamel in much older therapsid (ca. 263–241 Myr) teeth could not be distinguished from dentine. mCT is thus an accurate technique for measuring enamel thickness in recent taxa, although heavily mineralized teeth pose an obstacle to the ability of mCT to distinguish dental tissues. Moreover, absolutely thin enamel (less than ∼ 0.10 mm) is difficult to resolve adequately in raw mCT images based on pixel values alone. Therefore, caution must be exercised in the application of mCT to the study of fossilized teeth. © 2006 Wiley-Liss, Inc.