The role of protein kinase-C (PKC) in the potentiation of insulin release by arginine vasopressin (AVP) and acetylcholine (ACh) was investigated with normal mouse islets. The islets were submitted to a short term (30-min) or long term (22-h) treatment with phorbol 12-myristate 13-acetate (PMA) to stimulate acutely or down-regulate PKC before being stimulated by AVP or ACh. Control islets were treated with the inactive 4 alpha-phorbol 12,13-didecanoate. In the presence of 15 mM glucose and 2.5 mM Ca2+, AVP and ACh stimulated inositol phosphate (IP) formation, increased cytoplasmic Ca2+ (Cai2+), and potentiated insulin release. These effects were greater with ACh than with AVP, in particular on Cai2+, which was scarcely affected by AVP. In the absence of extracellular Ca2+, only ACh induced a short-lived increase in Cai2+ and insulin. Acute treatment with PMA in the presence of extracellular Ca2+ strongly increased insulin release in spite of a marked lowering of Cai2+. Under these conditions, the effects of AVP and ACh on IP production and Cai2+ were practically abolished, and only ACh transiently increased insulin release. In the absence of Ca2+, the small mobilization of Cai2+ by ACh triggered a peak of insulin, whereas a similar mobilization of Cai2+ by AVP was ineffective on insulin. After long term treatment of the islets with PMA, AVP normally increased IP formation, but did not affect insulin release. The effect of ACh on IP was still inhibited. However, ACh produced a marked transient increase in Cai2+, with a small transient release of insulin. The releasing effect of ACh was also inhibited in the absence of Ca2+. In conclusion, PKC plays a dual role in the B-cell responses to ACh and AVP. Its activation is necessary for the sustained potentiation of insulin release that both agents produce. This effect probably results from a sensitization of the secretory machinery to Cai2+, the triggering signal. PKC also exerts a negative feedback control on the signal transduction mechanisms involving phospholipase-C, but the ACh and AVP responses are not equally affected by this feedback.