Phorbol esters cause long term activation of protein kinase C (PKC) and frequently the down-regulation of PKC protein levels in mammalian cells. Mammalian PKC-gamma, -delta, and -eta down-regulated in response to phorbol esters when expressed in Schizosaccharomyces pombe. However, PKC-epsilon does not down-regulate in S. pombe, in contrast to the behavior of this isotype in mammalian cells. Co-expression of PKC-gamma or -delta with PKC-epsilon in S. pombe renders PKC-epsilon susceptible to down-regulation. A protein kinase defective form of PKC-delta does not down-regulate efficiently in S. pombe but, like PKC-epsilon, is susceptible when co-expressed with PKC-gamma or full-length PKC-delta. Thus, down-regulation is a consequence of the catalytic function of certain PKC isotypes with other isotypes being affected in trans. PKC down-regulation parallels a striking accumulation of vesicles in S. pombe, suggesting a direct relationship between these events.