The effects of cyproheptadine (CPH) added in vitro were studied in rat pancreatic islets maintained in culture medium. CPH added over 6 days resulted in either an increase (5 X 10(-7) M CPH) or a marked, but reversible decrease (5 X 10(-5 M) in insulin content of islets when related to that of controls. At both concentrations, however, total recoverable insulin from islets, cells detached from islets, and medium was decreased relative to control cultures. The increased insulin content observed after 6 days with 5 X 10(-7) M CPH may be explained by the partial inhibition of insulin release, preventing the normally occurring early drop in insulin content of control islets. The decreased total recoverable insulin in the culture system with 5 X 10(-5) M CPH (17% of the initial insulin content of the islets placed into the CPH-containing culture medium) was not acounted for by the combined effects of insulin degradation in the culture medium and inhibition of insulin biosynthesis. Together and by exclusion these data suggest increased insulin degradation within beta-cells as a result of exposure to 5 X 10(-5) M CPH. Since increased intracellular insulin degradation was not found at 5 X 10(-7) M CPH, the data suggest that only severe inhibition of insulin release (5 X 10(-5) M CPH) increases intracellular insulin degradation. CPH added in vitro irreversibly decreased islet glucagon content; the data suggest that these effects are due to alterations in the physical properties of the peripheral cell layers of isolated islets. Studies with 5 X 10(-5) M CPH on the biosynthesis of insulin immunoreactive material failed to link the appearance of flocculent material in dilated cisternae of the rough endoplasmic reticulum (observed by electron microscopy) with accumulation of an immunoreactive biosynthetic precursor for insulin.