Urokinase-type plasminogen activator (u-PA) activity was found in the medium as well as in the lysates of cultured uninduced Friend leukemia (FL) cells. PA activity progressively increased during the cell differentiation induced by dimethyl sulphoxide (DMSO), 4-hydroxy-3-methoxybenzaldehyde or hypoxanthine. Both the differentiation and the enhancement of PA activity in cultures of DMSO-induced cells were blocked by treating the cells with 1 microM dexamethasone. A highly significant correlation (rs = 0.93) was found between the number of hemoglobinized cells and the rate of PA secretion, indicating that the increase in PA activity coincides with late events of the differentiation process. FL cells specifically adhere to fibronectin-coated surfaces but tend to lose this property during the differentiation process. Anti-u-PA IgG antibodies promoted the attachment of differentiating cells to fibronectin-coated surfaces, suggesting that u-PA plays a role in the detachment of FL cells from fibronectin immobilized on the growth substratum.