Adult mouse pancreatic acinar cells were cultured as monolayers on collagen gels. Cells plated in medium containing 10% fetal bovine serum (FBS), epidermal growth factor (1 nM), carbachol (1 microM), insulin (1 microM), and corticosterone (10 nM) showed adaptive and growth responses. An adaptive phase occurred over the first 4-5 days, during which there was an approximately 50% decrease in the content of protein and DNA in the cultures, and the remaining attached cells showed reduced contents of zymogen granules. Subsequently, the cells spread out, divided, and formed confluent monolayers by days 11-14. Cell division was indicated by a doubling of the content of protein and DNA between days 5 and 9 and a 13-fold increase in thymidine incorporation. Microscopic examination of 14-day cultures revealed monolayers of cuboidal cells with morphological features of pancreatic acinar cells. To determine the direct effects of cholecystokinin on pancreatic acinar cell growth, cells were cultivated in media lacking added hormones and containing only 2.5% FBS with or without caerulein, a cholecystokinin analogue. Caerulein led to a 152% increase in DNA and an 89% increase in protein at day 9. By use of a preincubation [3H]thymidine incorporation assay, caerulein induced a dose-dependent increase in [3H]thymidine incorporation and nuclear labeling index, which was detectable at 0.1 nM, one-half maximal at 1 nM, and a maximal threefold increase occurred at 10 nM concentration. The results demonstrate a direct effect of caerulein on the pancreas to induce cell growth.