Abstract The phosphodiesterase inhibitor 3-isobutyl-l-methylxanthine (IBMX) promoted the formation of monolayers in cultured pancreatic islets isolated from neonatal rats. Immunofluorescence with specific antisera to insulin and glucagon revealed B-cells and A-cells in these monolayers. Glucose-mediated insulin release was increased by raising the glucose concentration from 5 to 10 mmoles/1. Addition of IBMX (0.1 mmoles/1) to medium containing 10 moles/I glucose produced a further increase in insulin release. Recovery of total insulin i.e. intracellular insulin plus insulin secreted, was also increased by ~50% after 8 days of culture. The B-cells showed a marked biosynthetic response to an acute glucose challenge after prior culture with 10 mmoles/1 glucose. Although both unstimulated (1.5 mmoles/1 glucose) and stimulated rates (15 mmnoles/1 glucose) of [ 3H]leucine incorporation into (pro)insulin were significantly higher following culture in 10 mmoles/1 glucose plus IBMX (0.1 mmoles/1) than after prior culture with 10 mmoles/1 glucose alone, the percentage of (pro)insulin synthesized in relation to total protein synthesis was only increased at the low concentration of glucose. These studies demonstrate that monolayer cultures Of neonatal B-cells can be readily produced by IBMX and maintained in a functional state, as defined by their secretory and biosynthetic response. It is suggested that the phosphodiesterase inhibitor exerts a sensitizing effect on the responsiveness of the B-cell to glucose. Moreover, the culture system employed in the present study may prove to be useful for further studies of various agents affecting the B-cell function.