Before weaning, arginine biosynthesis from citrulline most likely takes place in the small intestine rather than in the kidney. We studied the expression of ornithine cycle enzymes in the rat small intestine during perinatal development. The spatiotemporal patterns of expression of ornithine aminotransferase, carbamoylphosphate synthetase, ornithine transcarbamoylase, argininosuccinate synthetase, argininosuccinate lyase, and arginase mRNAs were studied by Northern blot analysis and in situ hybridization. In addition, the expression of carbamoylphosphate synthetase and argininosuccinate synthetase protein was studied by immunohistochemistry. Before birth, the developmentally more mature proximal loops of the intestine expressed the mRNAs at higher concentrations than the more distal loops. After birth, this difference was no longer obvious. The mRNAs of argininosuccinate synthetase and argininosuccinate lyase, the enzymes that metabolize citrulline to arginine, were detectable only in the upper part of the villi, whereas the other mRNAs were concentrated in the crypts. The distribution of argininosuccinate synthetase protein corresponded with that of the mRNA, whereas carbamoylphosphate synthetase protein was present in all enterocytes of the crypts and villi. Hepatic arginase mRNA could not be detected in the enterocytes. The spatial distribution of the respective mRNAs and proteins along the villus axis of the suckling small intestine indicates that the basal enterocytes synthesize citrulline, whereas the enterocytes in the upper half of the villus synthesize arginine.