The involvement of the phosphatidylinositol 3-kinase pathway in the regulation of melanogenesis was examined using human G361 melanoma cells. In the cells treated with wortmannin, a potent inhibitor of phosphatidylinositol 3-kinase, the melanin content increased concomitant with the elevated protein level of tyrosinase, a key enzyme in melanogenesis. Northern blot analysis revealed that the mRNA level of tyrosinase increased transiently on treatment of the cells with the phosphatidylinositol 3-kinase inhibitor. When the cells were infected with the adenovirus vector encoding the mutant adapter subunit of phosphatidylinositol 3-kinase, which acts as a dominant negative of phosphatidylinositol 3-kinase, both the melanin content and the expression of tyrosinase increased. In cells infected with the adenovirus vector encoding the constitutively active mutant of the lipid kinase, a decrease in melanin content as well as reduced expression of tyrosinase was observed. In cells expressing the constitutively active mutant of the serine-threonine protein kinase Akt, one of the downstream targets of phosphatidylinositol 3-kinase, the melanin content decreased as in the cells overproducing the constitutively active mutant of phosphatidylinositol 3-kinase. These results indicate that phosphatidylinositol 3-kinase regulates melanogenesis by modulating the expression of tyrosinase, and that activation of Akt is sufficient for suppression of melanin production in G361 melanoma cells.