The intraocular pressure-lowering drug latanoprost, a phenyl-substituted analogue of prostaglandin F2 alpha (PGF2 alpha), increases iris pigmentation in a small number of patients. In theory, this could be due to increased melanogenesis or melanocyte proliferation. To distinguish these two possibilities, the present study examined the effects of latanoprost on tyrosinase activity (the rate-limiting step for melanin synthesis) and mitotic index of cultured melanoma lines. Murine cutaneous melanoma lines (S91 and B16), and human uveal (OCM1, OCM3, and OM431) and cutaneous (SK-MEL5 and M21) melanoma lines were cultured with PGE1, PGE2, PGF2 alpha, latanoprost, or the adenylate cyclase stimulating agent forskolin. After treatment, tyrosinase was assayed with respect to its dopa oxidase activity using a colorimetric assay. PGE1, PGE2, PGF2 alpha, and latanoprost greatly increased tyrosinase activity in murine melanoma lines and caused small increases in tyrosinase activity in human uveal and cutaneous melanoma lines. Similar results were obtained with the cAMP-elevating compound forskolin. Cyclic AMP content, as determined by an enzyme-linked immunoassay, was similarly increased by all treatments, with forskolin being the most potent stimulator. Since the species difference in tyrosinase activity was observed without an apparent difference in induction of cAMP, latanoprost would appear to induce tyrosinase activity through a non-cAMP-dependent pathway. Finally, latanoprost and PGF2 alpha did not enhance the mitotic index of human uveal or cutaneous melanoma lines, measured by [6-3H] thymidine uptake, although they increased the mitotic index of one murine cutaneous line. Given that latanoprost induced tyrosinase activity, but did not increase the mitotic index in any of the human melanoma lines studied, this suggests that the in vivo iris pigmentation side effect of latanoprost may not result from increased cell division, but from elevated tyrosinase activity.