Glucagon is secreted from pancreatic a-cells in response to low glucose, and it is under neuronal and hormonal control. Protein kinase C (PKC) exists in different isoforms and it has been shown to play a crucial role in exocytosis in several cell-types. We aimed to investigate if PKC can modulate secretion in mouse and human a-cells and if so identify the specific isoforms involved. To this end we measured glucagon secretion by RIA, PKC isoform localization by confocal immunocytochemistry, and single-cell exocytosis as capacitance increases. In presence of 1 mM glucose glucagon secretion was stimulated 5-fold by the PKC-activator phorbol 12-myristate 13-acetate (PMA, 1mM; n=5; P<0.01) in mouse islets. A similar 6-fold stimulation (n=22; P<0.001) was observed in human islets. Interestingly, under the same conditions the PKC-antagonist bisindolylmalemide (BIM; 2.4 mM) inhibited glucagon secretion in mouse (n=6; P<0.01) but not in human (n=23). The stimulation of glucagon secretion by PMA was accompanied by a movement of PKCa and PKCd from the cytosol to plasma membrane (PM) in human a-cells. In mouse samples PKCa was already present at the PM prior to stimulation whereas PKCd was translocated to the PM by PMA. This suggests that PKCa is tonically active in mouse, but not in human a-cells. This was confirmed by capacitance measurements in mouse a-cells where a fraction of the a-cells had an amplified response already under control conditions, which could be reduced in the presence of BIM (n=5; P<0.01). Conversely, in the other fraction exocytosis was not affected by BIM, but stimulated by PMA (n=4; P<0.001). We conclude that both PKCa and PKCd are involved in PKC-stimulated glucagon secretion. In human a-cells PMA activates both isoforms, whereas in mouse a-cells PKCa is tonically activate and PMA primarily acts on PKCa.