Vitamin B-6 is important for skin development and maintenance. We examined vitamin B-6 metabolism in human and mouse skin collected at different phases of the hair cycle; in hamster melanomas; in normal and immortalized human keratinocytes (HaCaT) and several human melanoma cell lines. Pyridoxamine 5'-phosphate content was higher in mouse and hamster than in human skin. Activity of both pyridoxamine 5'-phosphate oxidase and pyridoxal 5'-phosphate hydrolase was significantly increased in rapidly growing melanomas compared to either normal skin or slower growing skin tumors. Reducing the pyridoxine content of the culture medium significantly increased the activity of pyridoxal kinase and pyridoxamine 5'-phosphate oxidase. Pyridoxal 5'-phosphate hydrolase has been proposed as a regulatory enzyme for vitamin B-6, but we found B-6 vitamer content to be significantly correlated only with kinase and oxidase activity and not with pyridoxal 5'-phosphate hydrolase activity. Although pyridoxal 5'-phosphate hydrolase activity is usually attributed to tissue-nonspecific alkaline phosphatase, tissue-nonspecific alkaline phosphatase knockout mice showed preservation of normal histology of the skin and adnexal structures. Furthermore, expression of tissue-nonspecific alkaline phosphatase mRNA was not detected in either HaCaT cells or human skin, both of which exhibited significant pyridoxal 5'-phosphate hydrolase activity. This suggests that an enzyme different from the classical tissue-nonspecific alkaline phosphatase may perform cutaneous pyridoxal 5'-phosphate hydrolase activity.