It has been reported that EGF treatment enhances uPA but not tPA in the A431 epidermoid carcinoma cell line. To determine whether the absence of tPA modulation by EGF could be due to the action of inhibitors, we assayed tPA, PAI-1, PAI-2 and tPA/PAI-1 complexes by immunological assays and zymography in A431 serum-free medium. We found that, under conditions in which EGF had no effect on tPA activity, tPA antigen increased with a concomitant rise of tPA/PAI-1 complexes, indicating the action of an inhibitor. Both tPA antigen and tPA/PAI-1 complexes were modulated by EGF in a time and concentration dependent manner. tPA/PAI-1 complex levels were lower than tPA levels, suggesting the presence of other inhibitors. Immunological assays detected PAI-2 in addition to PAI-1 and showed a time and dose response to EGF. Modulation of tPA and the anti-activators by the growth factor was confirmed by identification of the corresponding transcripts with cDNA probes. We conclude that the net plasminogen activator activity in A431 cells is the result of a balance between activators and inhibitors.