To document regional structural and cellular proliferation changes in the developing mouse colon, tissues from fetal, sukling, and weanling mice were analyzed by light microscopy (LM), transmission electron microscopy (TEM), scanning electron microscopy (SEM), [3H]-thymidine incorporation studies, and radioautography. The proximal and distal colon were studied independently at all ages. At 17–18 days of gestation, the mouse proximal colonic mucosa was projected into high and low longitudinal folds disposed in a V-shaped pattern. From birth up to 9 days, the mucosal folds observed by SEM can easily be misinterpreted as being a succession of high and low villus-like structures at LM level. TEM study confirmed the presence of highly specialized absorptive cells in the upper halves of the mucosal folds during this period. No recognizable crypts were noted at birth. Instead, LM and radioautography showed the presence of cell aggregates developing at the base of the epithelium at all levels of the mucosal folds. These cell aggregates evolved into rudimentary crypts giving fully differentiated crypts by day 16 with radiolabeled cells located in the midcrypt portion. As opposed to the proximal segment, a flat mucosa interspersed with well defined short crypts at birth was observed in the distal colon. During the following days, crypts further developed and by 16 days, the radiolabeled epithelial cells were still exclusively located at the base of the crypt. TEM observations illustrated that specialized cells as those found in the proximal segment did not differentiate in this segment. From birth up to 30 days, the labeling indices continuously decreased in the external muscle layer while increasing in the crypt epithelium at different time intervals in both colonic segments. The results show that true villus structures do not develop in proximal colonic mucosa and document regionally related morphological and cellular proliferation changes during mouse colonic maturation. © 1994 Wiley-Liss, Inc.