The regulation of plasmin, miniplasmin, and streptokinase-plasmin complex (SkPm) was studied in vitro in the presence of unfractionated porcine intestinal heparin using purified plasma proteinase inhibitors. Heparin enhanced the reaction of antithrombin III (AT) with plasmin (up to 40-fold with 20 units/ml). The rate of plasmin inhibition by alpha 2-antiplasmin (alpha 2AP) and by alpha 2-macroglobulin (alpha 2M) was not changed by heparin (0.5-100 units/ml); the rank-order of plasmin-inhibitory activity remained alpha 2AP greater than alpha 2M greater than AT. The reaction of miniplasmin with AT was studied also. The second order rate constant was 9.2 x 10(2) M-1s-1 without heparin and 2.6 x 10(4) M-1s-1 in the presence of 20 units/ml heparin. Heparin did not affect the rank-order of miniplasmin-inhibitory activity; it remained alpha 2M greater than alpha 2AP greater than AT. While the reaction of AT with SkPm was negligible, heparin stimulated this reaction dramatically. The SkPm-inhibitory activity of alpha 2AP was not changed by heparin. When plasma concentrations of alpha 2AP (1.05 microM) and AT (4.76 microM) were compared, AT inhibited greater amounts of SkPm in the presence of more than 5 units/ml of heparin. The increased SkPm-inhibitory activity of AT in heparin did not result from SkPm dissociation, and heparin did not decrease the rapid rate of streptokinase association with plasmin. These studies demonstrate that heparin can affect the regulation of fibrinolysis at multiple levels of the enzyme cascade.