Technical Abstract: The objective was to isolate enterocytes along the crypt-villus axis from the small intestine in the neonatal pig with the distended sac method. Alkaline phosphatase, aminopeptidase N, lactase and sucrase are marker enzymes for villus enterocytes, whereas in vivo 3H-thymidine incorporation into DNA is a marker for crypt enterocytes. Twelve hours after an intraperitoneal injection of 3H-thymidine (0.5 mCi/kg body weight), four segments (60 cm each) of proximal jejunum were dissected from each of 4 piglets (age 15-16 d) and were flushed with phosphate-buffered saline with 0.2 mM phenylmethylsulfonyl fluoride (PMSF) and 0.5 mM dithiothreitol (DTT). The segments were filled with oxygenated buffer with 27 mM sodium, 0.2mM PMSF and 0.5mM DTT and incubated in saline at 37 degrees C for 15 min. Twelve cell fractions (F1-F12, six 10-min and six 15-min fractions) were sequentially collected by incubating with oxygenated isolation buffer containing 1.5mM Na2EDTA, 0.2 mM PMSF and 0.5 mM DTT. 3H-thymidine incorporation in F9-F12 were about 7- to 10-fold higher than in F1-F4. Specific activities of marker enzymes decreased 5- to 10-fold between F1 and F12. The proportions of wet cell mass collected in F1-F4 (upper villus), F5-F8 (middle villus) and F9-F12 (crypt region) were 41.8, 45.4 and 12.8%, respectively. These results suggest that the distended sac method can be used to obtain enriched populations of mature villus and undifferentiated crypt enterocytes from neonatal pigs.