To investigate the beta-cell cytotrophic action of tolbutamide, pancreatic cells were dissociated from neonatal rat pancreata and then cultured for 10 days in the presence of various concentrations of tolbutamide. After counting insulin-positive beta cells, dose-response curves were plotted and analyzed. Increasing concentrations of tolbutamide cause an increase in beta-cell numbers until a maximum response is reached at a concentration of about 100 [mu] g/ml. At greater concentrations, tolbutamide becomes inhibitory. At concentrations of 100 [mu] g/ml of tolbutamide, beta-cell numbers are doubled. We further investigated the effect of glucose on tolbutamide-induced stimulation. Tolbutamide fails to stimulate beta cells after culture in medium containing low glucose concentrations. High concentrations of glucose are required for tolbutamide action. Preexposure for as little as 16 h to low glucose abolishes the stimulatory action of tolbutamide. The experiments indicate that adequate glucose concentrations are necessary for the preservation of tolbutamide-induced beta-cell survival and proliferation. Addition to the culture medium of the calcium channel blocker diltiazem or the calmodulin antagonist chlorpromazine affects a decrease in beta-cell numbers. This decrease depends on the concentration of the drug. Addition of tolbutamide reverses that decrease, which suggests that calcium is required for beta-cell survival and proliferation. Tolbutamide action seems to be specific, since fibroblasts are unaffected at concentrations of tolbutamide that are stimulatory for beta cells. At high concentrations, tolbutamide is cytotoxic for fibroblasts. Comparison with other sulfonylureas supports the superior activity of tolbutamide. The" second generation" sulfonylurea glyburide is cytotoxic. The metabolically inactive analog of tolbutamide, carboxytolbutamide does not stimulate beta cells, and tolazamide likewise has no statistically significant effect on beta cells. We conclude that pancreatic beta cells proliferate in culture in response to precise concentrations of nutrients and growth factors. Tolbutamide is a potent factor stimulating calcium-mediated beta-cell survival and proliferation.