The arachidonic acid lipoxygenase products released into culture supernatants by the human myeloid cell line K562 were quantitated by high-performance liquid chromatography. During 2 hr of incubation, K562 cells spontaneously released a mean of 9 ng of 15-monohydroxyeicosatetraenoic acid, 35 ng of 5-monohydroxyeicosatetraenoic acid, and 87 ng of a partially resolved mixture of 11- and 12-monohydroxyeicosatetraenoic acid per 10(7) cells. The addition of 50 micrograms of arachidonic acid per ml to the cell cultures increased the quantities of monohydroxyeicosatetraenoic acids generated after 2 hr of incubation by 10- to 100-fold; changes in the ratios of these lipoxygenase products occurred over 24 hr of incubation. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate, at concentrations of 5 to 25 ng/ml increased arachidonic acid lipoxygenation 1- to 2-fold in cultures containing 50 micrograms of arachidonic acid per ml, and up to 20-fold in cultures not supplemented with arachidonic acid. The lipoxygenation of arachidonic acid was enhanced 2- to 9-fold for up to 24 hr after a 2-hr exposure of K562 cells to 12-O-tetradecanoylphorbol-13-acetate. Nordihydroguaiaretic acid, a lipoxygenase inhibitor, blocked the effects of 12-O-tetradecanoylphorbol-13-acetate, suggesting that the monohydroxyeicosatetraenoic acids are the products of specific lipoxygenases rather than of nonenzymatic oxidative reactions. The capacities of phorbol esters to promote tumors in the mouse skin model corresponded to their respective capacities to enhance the lipoxygenation of arachidonic acid to 15- and 5-monohydroxyeico-satetraenoic acid but not to 11- and 12-monohydroxyeico-satetraenoic acid.