1. Measurements of cell capacitance were used to investigate the molecular mechanisms by which somatostatin inhibits Ca(2+)-induced exocytosis in single rat glucagon-secreting pancreatic alpha-cells. 2. Somatostatin decreased the exocytotic responses elicited by voltage-clamp depolarisations by 80 % in the presence of cyclic AMP-elevating agents such as isoprenaline and forskolin. Inhibition was time dependent and half-maximal within 22 s. 3. The inhibitory action of somatostatin was concentration dependent with an IC(50) of 68 nM and prevented by pretreatment of the cells with pertussis toxin. The latter effect was mimicked by intracellular dialysis with specific antibodies to G(i1/2) and by antisense oligonucleotides against G proteins of the subtype G(i2). 4. Somatostatin lacked inhibitory action when applied in the absence of forskolin or in the presence of the L-type Ca(2+) channel blocker nifedipine. The size of the omega-conotoxin-sensitive and forskolin-independent component of exocytosis was limited to 60 fF. By contrast, somatostatin abolished L-type Ca(2+) channel-dependent exocytosis in alpha-cells exposed to forskolin. The magnitude of the latter pool amounted to 230 fF. 5. The inhibitory effect of somatostatin on exocytosis was mediated by activation of the serine/threonine protein phosphatase calcineurin and was prevented by pretreatment with cyclosporin A and deltamethrin or intracellularly applied calcineurin autoinhibitory peptide. Experiments using the stable ATP analogue AMP-PCP indicate that somatostatin acts by depriming of granules. 6. We propose that somatostatin receptors associate with L-type Ca(2+) channels and couple to G(i2) proteins leading to a localised activation of calcineurin and depriming of secretory granules situated close to the L-type Ca(2+) channels.