Distribution of ornithine decarboxylase activity in rat intestinal villi and crypts was determined by serially sectioning frozen mucosa and measuring enzyme activity in pools of sections composed of villi or crypts. Contents of the pools was determined by histological examination of representative sections, and simultaneous measurement of sucrase as a marker of villus samples demonstrated excellent separation of villi and crypts. In fasted and ad lib fed rats, enzyme activity was highest in the villus-crypt junctional area and in crypts (P < 0.05). Refeeding after a fast increased enzyme activity 15-fold, with greatest activity in villus tips and the villus-crypt junctional area. Luminal 0.4 M glycine stimulated enzyme activity only in villus and villus-crypt junctional samples, while luminal 10 mM putrescine stimulated activity only in crypts. Parenteral epidermal growth factor caused increased enzyme activity in all mucosal areas, but the 18-28-fold increase in the three villus samples (top, middle and bottom) was significantly greater (P < 0.05) than the 7-9-fold increase in crypt and junctional samples. In rats refed after a fast, parenteral putrescine (2 mmol/kg) depressed enzyme activity in all mucosal areas. Ornithine decarboxylase activity is usually greatest in junctional and crypt cells, and villus and crypt cells respond differently to luminal and systemic stimuli.