Glu-plasminogen (Glu-plg), Lys-plg and Val442-plg (mini-plg) were activated by urokinase (UK), streptokinase (SK) or tissue plasminogen activator (t-PA). Their activation rates were kinetically analyzed. UK activated Lys-plg with smaller Km and nearly identical Vmax as Glu-plg. Mini-plg was activated by UK with higher Vmax but with the same Km as Glu-plg. On the other hand, t-PA activated Glu-plg, Lys-plg and mini-plg with the same Vmax, but Km was in the order of Glu-plg greater than mini-plg greater than Lys-plg. Fibrin hardly enhanced the UK activation of mini-plg, and fibrinogen, fragment D or E did not enhance the activation of mini-plg by UK. Only D domain, but not E domain, complexed with K5 of mini-plg and SK hydrolysed S-2251 faster than a dimolecular complex of mini-plg and SK, thus a trimolecular mixture of D, SK and mini-plg having a better activator activity than SK-mini-plg complex. In the t-PA activation of mini-plg, fibrin and fibrinogen enhanced the activation, but fragment D or E was ineffective, suggesting that a molecule containing more than one domain was required for the enhanced activation of mini-plg by t-PA.