Recent studies have provided evidence that apoptosis of pancreatic beta-cells is important in the early etiology of both type I and type II diabetes mellitus. The mechanisms responsible for induction of apoptosis are unknown, but we present evidence that the signal transduction pathway controlling the process in pancreatic beta-cells is regulated by G-proteins. We have employed the global G-protein activator fluoride and show that this agent induces apoptosis in clonal RINm5F pancreatic beta-cells and also in the cells of normal rat islets of Langerhans. The process is time and concentration dependent and may reflect the formation of AIF4- since it was inhibited by the aluminum chelator deferoxamine. Induction of apoptosis by fluoride was confirmed by acridine orange staining of cell nuclei, by electron-microscopic examination of chromatin condensation, and by oligonucleosomal degradation of DNA. The involvement of G-proteins was confirmed by culture of beta-cells in the presence of pertussis toxin (PTX) prior to exposure to fluoride. PTX did not affect the extent of cell death under control conditions but it consistently, and markedly, enhanced the response to fluoride. The results demonstrate that apoptosis can be induced in pancreatic beta-cells by sustained activation of a G-protein-dependent signaling pathway(s) and they further suggest that a pertussis toxin-sensitive G-protein is involved in attenuation of the response. Treatment of RINm5F pancreatic beta-cells with dibutyrylcAMP resulted in a dose-dependent, saturable increase in cell death, suggesting that a sustained rise in intracellular cAMP may form part of the effector system controlling apoptosis.