Proliferating cells have a restricted three-dimensional spatial distribution within the crypt, which is the proliferative unit of the colon. Accurate quantitative and spatial analyses of S phase cells in the colon have therefore been limited by histological techniques. To overcome these limitations, S phase cells in microdissected intact colonic crypts of control, modified-starved, and refed rats were labeled by histone H3 in situ hybridization and analyzed by confocal microscopy. High-resolution digital images of the crypt cell nuclei stained with cyanine nucleic acid and of the labeled S phase cells were produced from confocal microscopic optical crypt sections. The S phase labeling index (LI) per whole crypt significantly (P < 0.001) discriminated the proliferative differences between control, modified-starved, and refed rats and correlated (r = 0.92) with the LI determined from histological crypt sections of the same rats. The variance component of the LI attributable to differences between whole crypts, 0.44 (95% confidence interval, 0.38-0.51), was considerably smaller than that attributable to differences between histological crypt sections, 6.07 (95% confidence interval, 5.18-6.96). Confocal microscopy and histone H3 in situ hybridization of intact three-dimensional crypts enables precise in vitro quantitation and spatial analysis of the total and S phase crypt cells.