The purpose of this study was to investigate the effects of two pyrimidine antimetabolites, arabinosyl cytosine (ara-C) and 5-azacytidine (5-aza-C), on the intestinal transport of glucose, amino acids, and electrolytes. Male Swiss-Webster mice were given either 50 mg/kg ara-C or 15 mg/kg 5-aza-C in isotonic saline, ip, once daily for 5 successive days. Using the everted sac technique there was a marked decrease in d-glucose, 3- O-methyl- d-glucose (3MG), and l-tyrosine transport. The rates of glucose, 3MG, and l-tyrosine transport were decreased by 67, 74, and 60%, respectively, in ara-C-treated animals, and 58, 79, and 62%, respectively, in 5-aza-C-treated animals. When intestinal sacs from untreated animals were exposed to ara-C or 5-aza-C (10 −3 m) on both mucosal and serosal sides, there was no effect on transport. Furthermore, in animals treated with either ara-C or 5-aza-C there was a significant decrease in transmucosal potential difference and short circuit current associated with decreases in net Na +, Cl −, and HCO 3 − transport across the intestinal mucosa. Simultaneous administration of deoxycytidine, in a two- to threefold molar excess, prevented the impairment of intestinal transport function caused by ara-C; similarly, cytidine prevented the impairment caused by 5-aza-C. The results suggest that active metabolites of ara-C and 5-aza-C are involved in their inhibitory effects on intestinal absorption and that this may be a contributing factor to the malnutrition and diarrhea commonly associated with the administration of these anticancer drugs.