The effects of glucose on cAMP accumulation, insulin (IRI) release, and proinsulin synthesis were simultaneously measured in isolated rat islets. Proinsulin synthesis was assayed by measuring the incorporation of [3H] leudne into islet proteins reacting with Sepharose-bound anti-insulin gamma globulin. The accumulation of cAMP in islets and incubation medium was measured by the incorporation of [3H]-adenine into [3H] CAMP and purified by ion-exchange chromatography and barium sulfate precipitation. Glucose dose-response studies were performed in the absence and presence of 0.1 and 1.0 mM isobutylmethylxanthine (IBMX). The IRI and cAMP responses to glucose demonstrated similar stimulation thresholds and Km values at all IBMX concentrations. With increasing amounts of IBMX, the Vmax was increased and the Km decreased for both responses. There was parallelism between IRI secretion and accumulation of [3H] CAMP in the medium, except at nonstimulatory glucose concentrations, where, especially in the presence of 1.0 mM IBMX, a marked stimulation of cAMP accumulation was accompanied by only modest IRI release. Proinsulin biosynthesis showed a much greater sensitivity to glucose than the cAMP and IRI responses. The threshold for stimulation by glucose was around 1.4 mM, and the response already saturated at 7.0 mM. Except when 1.0 mM IBMX was present, no IRI or cAMP responses were registered in this glucose dose range. These findings indicate that IRI release and proinsulin synthesis have distinct glucose recognition mechanisms and that ghicoseinduced cAMP stimulation does not mediate the effect of the hexose on hormone synthesis in the islet cell. When islet cAMP was elevated with IBMX, a decrease in the Km of the proinsulin response to glucose was observed, while the Vmax of the reaction was only slightly modified. This suggests that the level of cAMP in the islet may modulate the sensitivity of the B-cell for the effect of glucose on insulin biosynthesis and thus provide a mechanism for the regulation of insulin biosynthesis by such factors as gut hormones and glucagon under in-vivo conditions.