We investigated the roles of calcium ion (Ca2+) on euglobulin clot lysis time (ECLT) and found that the physiological concentration of Ca2+ significantly (4-5 times) shortened ECLT. The shortening was observed at the concentration of Ca2+ higher than 1.5-2.0 mM. Other divalent cations such as Mg2+, Zn2+ or Mn2+ didn't change ECLT. Anti-tPA antibody or plasminogen activator inhibitor-1 prolonged ECLT in the absence of Ca2+, whereas they had no effect on ECLT shortened by Ca2+. C1 inactivator also had no effect. When barium absorbed plasma was employed, the shortening of ECLT by Ca2+ wasn't observed, whereas it was recovered by the readdition of barium absorbed fraction. When factor X deficient plasma was employed, the shortening of ECLT by Ca2+ was also not observed. Thus, Ca2+ enhances fibrinolysis in a cell free system by a novel pathway in which the presence of factor X is prerequisite.