In platelets and other secretory cells, protein kinase C (PKC) plays a role in exocytosis stimulated by physiological extracellular signals, although its linkage to the secretory machinery is poorly understood. We investigated whether Rab6, a GTP-binding protein that fractionates with platelet alpha-granules, may be involved in linking these processes. We found that Rab6 contains two PKC consensus phosphorylation sites that are evolutionarily conserved. In platelets metabolically labelled with [(32)P]P(i), Rab6 phosphorylation was induced by phorbol esters or by thrombin. This phosphorylation was blocked by a specific PKC inhibitor (Ro-31-8220), but not by a p38 mitogen-activated protein kinase inhibitor (PD-169316). Physiological stimulation of platelets caused a PKC-dependent translocation of Rab6 from platelet particulate fractions, nearly doubling the fraction of Rab6 in the cytosol. A human Rab6 isoform (Rab6C) that is preferentially expressed in human platelet RNA was cloned and its phosphorylation by PKC was characterized. Rab6C incorporated up to 2 mol of [(32)P]P(i) per mol of active protein. Rab6C bound GDP and GTP with K(d) values of 113+/-12 and 119+/-27 nM respectively, and hydrolysed GTP at a rate of 100+/-15 micromol of GTP/mol of Rab6C per min. PKC phosphorylation of Rab6C increased the affinity for GTP by 3-fold, although it had lesser effects on GDP (1.6-fold). Phosphorylation did not alter the GTPase activity. In summary, thrombin activation of platelets leads to PKC-dependent phosphorylation of Rab6 and a translocation of Rab6 to the cytosol. We suggest that PKC phosphorylation may be an important mechanism through which Rab functional interactions in vesicle trafficking and secretion can be altered in response to an external stimulus.