We have investigated the behavior of dissociated cells of a moderately differentiated Longnecker transplantable pancreatic acinar carcinoma (Longnecker et al., Cancer Lett., 7: 197-202, 1979) maintained in vitro on acellular seminiferous tubular basement membranes of rat testis. The tumor cells, which grow as solid masses in vivo with little organization, undergo organogenesis in vitro into distinct duct-like structures with lumina in the presence of basement membrane scaffoldings. These duct-like structures were formed by flattened epithelial cells, which exhibited poorly differentiated acinar cell characteristics with few or no zymogen (secretory) granules. The cells lining the duct-like structures retained the pancreatic acinar cell specific antigen as determined by indirect immunofluorescence. DNA synthesis did not accompany duct-like organization; however, all of the cells lining these structures continued to incorporate [3H]leucine for up to 4-5 days of culture. They continued to synthesize and secrete amylase, a marker protein of pancreatic acinar cells, into the medium. These results demonstrate that neoplastic epithelial cells of Longnecker pancreatic tumor differentiate into duct-like structures when they come into contact with a basement membrane scaffolding and do not accumulate well-formed secretory granules. This is in marked contrast to the previously reported in vitro differentiation of cells derived from another transplantable rat pancreatic acinar cell carcinoma where the neoplastic cells were fully cytodifferentiated on seminiferous tubular basement membrane without forming duct-like structures but accumulated abundant well-developed zymogen granules (Watanabe et al., Cancer Res., 44: 5361-5366, 1984). Although the basal lamina promotes differentiation of cells of two different pancreatic carcinomas in vitro, we conclude that the in vitro expression of morphogenetic and cytodifferentiation patterns is dependent upon the intrinsic properties of cells of these two transplantable pancreatic tumors.