The role of protein phosphorylation in MSH-induced melanogenesis was investigated with an in vivo phosphorylation assay using intact cultured Cloudman S91 mouse melanoma cells preincubated with [32P]orthophosphate. Exposure of the cells to α-MSH increased the extent of labelling of two protein bands on SDS gel electrophoresis with estimated molecular weights of 43 and 34 kDa, respectively. The 32P incorporation was concentration-dependent and reached a maximal value at 10-8 M α-MSH for the 43 kDa band (156% of controls) and at 10-5 M α-MSH for the 34 kDa band (250% of controls). The corresponding ED50s were 5 × 10-10 M (43 kDa) and 3 × 10-8 M (34 kDa). The 32P incorporation into the 34 kDa band reached a maximum after a 5 min exposure to α-MSH whereas 43 kDa phosphorylation was maximal after a 30-60 min incubation with hormone. The effect was completely reversible after removal of the hormone and specific for melanotropic peptides. Dibutyryl cAMP (103̄ M) and forskolin (10-4 M) together with isobutylmethylxanthine (10-4 M) mimicked the effect of α-MSH, pointing to an involvement of adenylate cyclase activation in the phosphorylation of both the 34 kDa and the 43 kDa protein. Preliminary observations showed that the 34 kDa protein is membrane-bound whereas the 43 kDa protein is of mitochondrial or melanosomal origin. © 1987.