Gamma-glutamyl transpeptidase (GGT), an enzyme of the gamma-glutamyl cycle, was demonstrated in 3 of 6 cell lines derived from a single B16 murine melanoma. Its activity in these cells varied a great deal, appeared to be correlated with the developmental cycle of the cells, and was greatest in young, actively melanogenic cells. Generally, the activity seemed parallel to that of tyrosinase, an enzyme specific for melanin synthesis. The levels of both enzymes tended to decline with prolonged in vitro cultivation, but could be readily renewed after one animal passage. The 3 cell lines that were GGT-negative were nonpigmented and DOPA-negative; so was a nonmelanogenic and nonmelanocytic rhesus cell line. Melanocyte-stimulating hormone (MSH) and theophylline both enhanced pigmentation in murine melanoma cells. The mechanisms of their action apparently differed. We found that theophylline increased both DOPA- and GGT- reactive cells, whereas MSH only increased DOPA-reactive cells. All 3 GGT-positive lines were tumorigenic, and 2 GGT-negative line were not tumorigenic. Our observations suggest that GGT plays a role in the melanin biosynthetic pathway and the its activity is greater in melanoma cells that are tumorigenic.