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Plasminogen activator (PA) activity of human promyelocytic leukemia cell line HL‐60 was assayed by following the conversion of plasminogen to plasmin and the plasmin‐mediated hydrolysis of 14C‐labeled globin. When HL‐60 cells were induced to differentiate into macrophages by 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA), cell‐associated PA activity and secretion of PA into the conditioned medium increased profoundly. PA activity increased earlier and as a result of lower concentrations of TPA than the ability of the cells to adhere. Exposure to 10−6M dexamethasone did not prevent TPA‐induced adherence and produced a slight inhibition of cellular PA activity. These findings imply that TPA‐induced differentiation of HL‐60 cells to macrophage‐like cells is associated with induction of PA activity.