An active fraction that accelerates plasminogen activation by tissue-type plasminogen activator (t-PA) was purified from a haemolysate of bovine erythrocytes. When the haemolysate was mixed with t-PA, it produced a 2- to 3-fold increase in plasminogen activation as measured by an insoluble fibrinolytic assay system and a soluble amidolytic assay system with the chromogenic substrate S-2251. Zymographic analysis showed that, while the haemolysate increased t-PA activity, it did not alter the electrophoretic characteristics of the t-PA nor did it induce any fibrinolysis in the absence of t-PA or plasminogen. The haemolysate was devoid of plasmin and plasminogen activator activity but was most effective in accelerating plasminogen activation by t-PA in the presence of substrate. Based on the purification characteristics of the active fraction in the haemolysate, it appears to have a molecular weight of less than 10 kDa.