It is well known that phospholipase D plays a crucial part in the signal transduction of many types of cells, and is activated by protein kinase C alpha when cells are stimulated. To elucidate the role of phospholipase D in melanoma, the expression of phospholipase D1 and protein kinase C alpha in primary and metastatic lesions of acral lentiginous melanoma and superficial spreading melanoma was investigated using immunohistologic techniques. In addition, the mechanism of regulation of phospholipase D1 by protein kinase C alpha was examined in a human melanoma cell line HM3KO using an adenovirus-mediated gene transfer technique. Both phospholipase D1 and protein kinase C alpha were strongly expressed in primary and metastatic lesions of superficial spreading melanoma. Conversely, in acral lentiginous melanoma lesions, the expression of these two proteins increased dramatically with tumor progression; the expression of both phospholipase D1 and protein kinase C alpha was almost negative in the radial growth phase of primary acral lentiginous melanoma lesions, and increased synchronously in a progression-related manner in advanced acral lentiginous melanoma lesions, including vertical growth phase and metastatic lesions. Immunoprecipitation study showed that phospholipase D1 and protein kinase C alpha are associated physiologically in resting melanoma cells. Further immunoprecipitation study using HM3KO cells after adenovirus-mediated simultaneous overexpression of phospholipase D1 and protein kinase C alpha, or phospholipase D1 and the kinase-negative mutant of protein kinase C alpha revealed that both protein kinase C alpha and the kinase-negative mutant of protein kinase C alpha are associated with phospholipase D1 in melanoma cells in the absence of an external signal. Overexpression of protein kinase C alpha or the kinase-negative mutant of protein kinase C alpha in melanoma cells by the adenovirus vectors resulted in the enhancement of basal phospholipase D activity in a viral concentration-dependent manner. Furthermore, enhanced basal phospholipase D activity increased the in vitro invasive potential of HM3KO cells. These results suggest that upregulation of phospholipase D1 and protein kinase C alpha plays a part in the progression of acral lentiginous melanoma from the radial growth phase to the vertical growth phase. The present results also suggest that protein kinase C alpha associates with phospholipase D1 and enhances basal phospholipase D activity in a protein phosphorylation-independent manner in melanoma cells, which contributes to the cell's high invasive potential.