Carbonic anhydrase activity was localized histochemically by light and electron microscopy in the proximal and distal colon of developing rats. Fixed tissue was taken for normal morphology and carbonic anhydrase localization from fetal (20-22 days gestation), suckling (1-19 days postnatal), weanling (20-25 days postnatal), and adult rats. The proximal colon had distinct villi at birth which were diminished between days 5 and 11 postnatally. The distal colon lacked villi at birth but had rudimentary crypts (ridges and furrows) which were replaced during the suckling period by a flat mucosa interspersed with true crypts. Carbonic anhydrase first appeared in both proximal and distal colonic epithelial cells on the day of birth (22 days gestation). Goblet cells were nonreactive at each developmental period. In neonatal rats, epithelial cells in the upper half of the villi of the proximal colon and on the surface and upper crypts of the distal colon were positive for carbonic anhydrase throughout the cytoplasm. Cells at the villar base (proximal colon) or in the deep crypt (distal colon) had reaction product in the intercellular spaces but not the cytoplasm. By 11 days postnatal, cytoplasmic reaction product was present in proximal colonic cells in the upper three-fourths of the crypt and was concentrated in a heavy band in the apical cytoplasm. In the distal colon, cytoplasmic positive cells did not extend as deeply into the crypts and the apical banding pattern was weak. Intercellular spaces in the deeper crypt epithelium were positive in both proximal colon and distal colon, suggesting a membrane-bound carbonic anhydrase. It was concluded that carbonic anhydrase appeared suddenly at birth and was continuously present in mid- to upper-crypt (or upper villus in early neonatal proximal colon) non-goblet cells into adulthood. This suggests a functional role for carbonic anhydrase in chloride-bicarbonate exchange across the neonatal and adult colonic mucosa.