Our objective was to examine the distribution of enterocyte digestive enzyme activity along the crypt-villus and longitudinal axes of the small intestine in formula-fed neonatal pigs between the ages of 14 and 18 d. The distended intestinal sac method was used to isolate 12 sequential fractions (F1 through F12) of epithelial cells. Enterocyte migration rate was measured in the proximal and distal intestine using in vivo bromodeoxyuridine labeling. Specific activities of representative villus cell marker enzymes of alkaline phosphatase, aminopeptidase N, sucrase, and lactase increased 6- to 17-fold from F12 (crypt cells) to F1 (villus cells), whereas the crypt cell marker [3H]thymidine incorporation increased 8- to 18-fold from F1 (villus cells) to F12 (crypt cells). Enterocyte migration rate was similar (3.2 vs 3.0 microm/h), whereas the villus height (547.4 vs 908.5 microm) and enterocyte life span (4.7 vs 10.2 d) were markedly lower (P < 0.05) in the proximal than in the distal segments, respectively. In general, the specific activities of all enzymes were lowest in the crypt fractions (F9 through F12) but increased markedly (ranging from 8- to 17-fold) from F12 to F1. The activity of aminopeptidase N was higher and that of sucrase was lower in the distal than in the proximal segment. The activities of the remaining enzymes were similar in the proximal and the distal segments. Our results suggest that the enterocyte life span in the distal small intestine is approximately twice as long as in the proximal small intestine. However, despite the difference in life span, the patterns of enzyme activities along the crypt-villus axis were generally similar in the proximal and the distal regions.