Exposure of human HL60 cells to dimethyl sulfoxide results in their differentiation to mature granulocyte-like cells that concomitantly acquire the capacity to synthesize leukotrienes. The appearance of 5-lipoxygenase mRNA during differentiation indicated that these cells provide a useful model system for the biosynthesis and regulation of 5-lipoxygenase. Immunoblot analysis of protein from differentiated HL60 cells detected a 78,000-Da species comigrating with 5-lipoxygenase purified from human peripheral blood leukocytes. Metabolic labeling studies indicated that both undifferentiated and differentiated HL60 cells synthesized 5-lipoxygenase; however, the differentiated cells incorporated approximately 4.4-fold more [35S]methionine into 5-lipoxygenase protein than did controls. In addition, the differentiated HL60 cells contained approximately 3.3-fold more 5-lipoxygenase enzyme activity than undifferentiated cells. Metabolic labeling studies failed to demonstrate any post-translational modifications of 5-lipoxygenase, including proteolysis, mannose glycosylation, myristic acid acylation, or phosphorylation. When differentiated HL60 cells were incubated with [35S]methionine for 4 versus 16 h, no difference was observed in the pattern of total radiolabeled supernatant protein; however, there was a significant increase in the incorporation of radioactivity into immunoprecipitable 5-lipoxygenase protein from cells labeled for 16 as compared with 4 h. Pulse-chase studies demonstrated that the t1/2 of 5-lipoxygenase in these cells is approximately 26 h. Activation of differentiated HL60 cells with Ca2+ ionophore A23187 resulted in the loss of 5-lipoxygenase protein and activity from the cytosol and the accumulation of inactive protein in a membrane fraction. Following ionophore stimulation, no augmentation in the rate of 5-lipoxygenase synthesis occurred in order to compensate for the loss of the translocated/inactive enzyme. Finally, additional 5-lipoxygenase was able to translocate to the membrane in response to subsequent ionophore challenges.