New insulin-secreting cell lines (INS-1 and INS-2) were established from cells isolated from an x-ray-induced rat transplantable insulinoma. The continuous growth of these cells was found to be dependent on the reducing agent 2-mercaptoethanol. Removal of this thiol compound caused a 15-fold drop in total cellular glutathione levels. These cells proliferated slowly (population doubling time about 100 h) and, in general, showed morphological characteristics typical of native beta-cells. Most cells stained positive for insulin and did not react with antibodies against the other islet hormones. The content of immunoreactive insulin was about 8 micrograms/10(6) cells, corresponding to 20% of the native beta-cell content. These cells synthesized both proinsulin I and II and displayed conversion rates of the two precursor hormones similar to those observed in rat islets. However, glucose failed to stimulate the rate of proinsulin biosynthesis. In static incubations, glucose stimulated insulin secretion from floating cell clusters or from attached cells. Under perifusion conditions, 10 mM but not 1 mM glucose enhanced secretion 2.2-fold. In the presence of forskolin and 3-isobutyl-1-methylxanthine, increase of glucose concentration from 2.8-20 mM caused a 4-fold enhancement of the rate of secretion. Glucose also depolarized INS-1 cells and raised the concentration of cytosolic Ca2+. This suggests that glucose is still capable of eliciting part of the ionic events at the plasma membrane, which leads to insulin secretion. The structural and functional characteristics of INS-1 cells remained unchanged over a period of 2 yr (about 80 passages). Although INS-2 cells have not been fully characterized, their insulin content was similar to that of INS-1 cells and they also remain partially sensitive to glucose as a secretagogue. INS-1 cells retain beta-cell surface antigens, as revealed by reactivity with the antigangloside monoclonal antibodies R2D6 and A2B5. These findings indicate that INS-1 cells have remained stable and retain a high degree of differentiation which should make them a suitable model for studying various aspects of beta-cell function.