Two-chain tissue plasminogen activator (t-PA) was found to be inactive in a coupled colorimetric assay for plasminogen activators, but a high level of activity was obtained in the presence of poly-D-lysine. This stimulated activity was strongly inhibited by minactivin, a urokinase inhibitor, but unstimulated enzyme could be shown to be unaffected by minactivin. In the presence of poly-D-lysine minactivin was a very successful competitive inhibitor of t-PA with respect to the substrate, plasminogen. The Ki for minactivin determined by the Henderson method was 2.5 X 10(-12) M, compared to the Km for plasminogen determined as 0.6 X 10(-6) M. The value of Ki for minactivin with u-PA, determined under the same conditions, was 1.6 X 10(-11) M.