Previous studies in rodent islets have suggested the existence of a small number of proliferating islet cells. Since islet tissue is composed of endocrine as well as nonendocrine cells, we examined whether the DNA synthesis that is detectable in intact islets in vitro corresponds to an activity of islet B cells, islet endocrine non-B cells and/or nonendocrine islet cells. DNA synthesis was quantified by [3H]thymidine incorporation in trichloracetic acid precipitable material, by nuclear thymidine labeling in autoradiographs, and by nuclear bromodeoxyuridine fluorescence. Adult islet endocrine purified B cells, as well as other endocrine islet cells, incorporated 1 to 2 fmol thymidine/1000 cells, which is 3 times lower than intact islet tissue and 30 times lower than nonendocrine islet cells. Addition of 10% fetal calf serum did not increase DNA synthesis in purified endocrine islet cells but doubled it in intact islets and enhanced it 8-fold in nonendocrine islet cells. The higher thymidine incorporation in intact islets was due to the presence of nonendocrine cells. An increase in medium glucose concentration from 100 to 200 mg/100 ml doubled the thymidine incorporation in purified islet B cells, but not in other endocrine islet cells; no concomitant increase in the number of thymidine or bromodeoxyuridine-labeled nuclei was observed. A phenomenon of glucose-stimulated DNA repair was not excluded. Using three different methods, we have found no evidence for a proliferating activity of adult rat islet B cells under the selected in vitro conditions of this study.