Retinoic acid treatment of B16 mouse melanoma cells induces a differentiated phenotype. This is accompanied by a decrease in monolayer growth rate, loss of the ability to form colonies in soft agarose, increased production of melanin and other melanocyte-specific markers. In addition, retinoic acid treatment of these cells decreases their tumorigenicity when injected subcutaneously into mice. Our laboratory has found that an early biochemical change after the addition of retinoic acid is a large increase in PKC. PKC is an enzyme whose activity is activated by diacylglycerol and calcium and has been shown to be an important mediator of substances that stimulate growth or differentiation. Since PKC is a multi-gene family, it was important for us to determine which isotype(s) was expressed in B16 cells and which type was induced by retinoic acid. We found that only PKC-alpha is expressed in these cells, and this is the form that is induced by retinoic acid. The retinoic acid-induced increased in PKC-alpha is found at both the RNA and protein level. The mechanism of induction is not yet clear since there is only a small increase in the transcription rate and no change in the stability of the mRNA for PKC-alpha in treated cells. In addition, the induction of PKC by retinoic acid can be blocked by inhibitors of protein synthesis, suggesting that the induction requires the synthesis of new protein(s). In order to determine the role of increased PKC-alpha in the retinoic acid-induced differentiation, we transfected full-length PKC-alpha cDNA in mammalian expression vectors into B16 cells. Two clones that stably overexpressed PKC-alpha to different levels were isolated. The phenotype of these clones resembled WT cells treated with retinoic acid, i.e. they had longer doubling times, decreased ability to form colonies in soft agar, increased melanin production, and decreased tumorigenicity in mice. Recent data suggest a role for the RAR-beta in mediating the effect of retinoic acid on PKC induction. B16 cells express a very low amount of RAR-beta mRNA. The level is increased drastically by retinoic acid treatment without any requirement for protein synthesis. When B16 cells were transfected with and overexpressed RAR-beta, they also expressed more PKC-alpha mRNA and protein, and the induction of PKC by retinoic acid was not blocked by protein synthesis inhibitors. In summary, these finding suggest a key role for PKC-alpha in the pathway by which retinoic acid induces B16 mouse melanoma differentiation.