The basal lysis rates ofex-vivoprepared blood clots from rats, mice, hamsters, dogs, and humans and levels of α2-antiplasmin (α2-AP) cross-linked to fibrin have been studied in the presence and absence of factor XIII (FXIII) inhibitors using a blood clot lysis assay and an α2-AP binding assay. Clots prepared from rat or human blood lysed spontaneously within 3-5h. Clots from hamster blood lysed completely within 30 min but clots prepared from murine or canine blood lysed only after addition of 1.0 IU human t-PA. To study the effect of activated FXIII (FXIII) inhibition on blood clot lysis the FXIII inhibitors L722151 and mono-dansylcadaverine (dansyl) were used. In the presence of the FXIII inhibitors human, murine, and canine blood clots showed increased lysis rates. The lysis rate of rat blood clots was not affected. Effects on hamster blood clots could not be detected because of the high basal lysis rate. In clots prepared from human, murine, or canine plasma about 20% of the plasma α2-AP concentration was cross-linked to fibrin. FXIII inhibitors inhibited crosslinking by more than 80%. No significant cross-linking of α2-AP could be detected in rat and hamster plasma clots. When 0.1 volume of human plasma was added to 0.9 volume of rat plasma the level of α2-AP cross-linking was equal to that in human plasma indicating that rat α2-AP can be cross-linked to human fibrin. The same effect was obtained with human FXIII deficient plasma but not with human fibrinogen deficient plasma. These experiments indicate that rat fibrinogen is not a substrate for the cross-linking of rat α2-AP catalysed by rat FXIII, Addition of human plasma to hamster plasma did not result in an increased level of α2-AP cross-linking which indicates that other factors are involved.