To determine the progenitor nature of centroacinar cells (CACs), we attempted to compare the expression pattern of endocrine cell markers and PDX-1 (pancreatic duodenal homeobox gene 1) in CACs of both the quiescent and the regenerating rat pancreas. In the normal pancreas, most CACs were relatively small cells with sparse cytoplasm and oval or elongated nuclei. In addition, we noticed a distinct population of a small number of large cells with round nuclei in the centroacinar region. By immunohistochemistry, 0.21% and 0.3% of CACs in normal rat pancreas were respectively found positive for glucagon and insulin, being large CACs and designated as GL-CAC and IL-CAC. They also exhibited the mRNA of each hormone by in situ hybridization (ISH). The ISH signal for glucagon but not insulin was also detected in a subset of small CACs (designated GS-CAC). The expression of PDX-1 was also observed in subsets of small and large CACs (PS-CAC and PL-CAC, respectively). After a 90% pancreatectomy, the relative frequency for GS-CACs, but not those for other CACs, was significantly reduced in two days after surgery. On day 7 after surgery, the number of GS-CACs recovered to preoperative levels, whereas GL-CACs, IL-CACs, PS-CAC, and PL-CAC gradually increased to about double in number. From these results, a portion of CACs was suggested to differentiated into endocrine cells. A possible cell lineage is discussed for endocrine neogenesis during pancreatic regeneration.