The proliferative capacity of individual immunocytochemically identified islet beta cells was investigated in tissue culture. The pancreatic digest was filtered through a 20-μm filter to eliminate partially digested tissue fragments and islets; it was then cultured at low density to allow assessment of single cells. The type of cell was identified immunocytochemically by reaction with antibody to insulin or glucagon, and DNA synthesis was estimated from autoradiographs after incorporation of tritiated thymidine. Single immunocytochemically reactive beta or alpha cells attached to the culture substratum and then proliferated, directly proportional to time in culture. Single beta cells did not incorporate thymidine after 1 day in culture; nonendocrine cells, presumed to be mainly fibroblasts, readily incorporated thymidine. Beyond the first day, about 10% of the beta cells incorporated thymidine. The number of radioactively labeled, immunocytochemically reactive beta cells increased for 5 days in culture and then remained at the elevated level until experiments were terminated at 12 days. DNA synthesis in beta cells occurred in two waves separated by about 3 days, suggesting a generation time of about 3 days for immunocytochemically identified beta cells. Fetal calf serum was found to be an essential culture medium ingredient to sustain thymidine incorporation; horse serum was found to be an unsuitable substitute. Mitotic figures were recorded in differentiated beta and alpha cells. These studies conclusively show that differentiated beta and alpha cells can proliferate in culture. An undifferentiated precursor cell type is not required to sustain endocrine cell proliferation.