We have studied the effects of lipoxygenase inhibition and metabolite addition of HL-60 cells induced to differentiate. When HL-60 are induced by dimethyl sulfoxide (DMSO) in the presence of an inhibitor of lipoxygenase, caffeic acid, there is a marked change from the expected phenotype of mature granulocytes to a population composed predominantly of mature monocytes. (DMSO alone: 54% granulocytes, 10% monocytes; DMSO + caffeic acid: 23% granulocytes, 53% monocytes.) Addition of leukotriene D4 to DMSO-induced, caffeic acid-inhibited cultures resulted in a dose-dependent recovery of the granulocyte phenotype. Addition of lipoxygenase inhibitors to phorbol ester-treated HL-60 cells did not alter the expected monocytic differentiation. These results support a role for leukotriene D4 in the regulation of granulocyte differentiation of HL-60 cells induced with DMSO.