Human factor XIII (FXIII) and tissue transglutaminase (tTG) are homologous proteins. FXIII requires thrombin for activation and cross-links the gamma chains of fibrin(ogen) more efficiently than the Aalpha chains. On the other hand, tTG is thrombin-independent and forms predominantly Aalpha and Aalpha-gamma chain complexes. Previous work from this laboratory demonstrated that amino acid residues within exon 7 of FXIII were important for catalysis (Hettasch, J. M., and Greenberg, C. S. (1994) J. Biol. Chem. 269, 28309-28313). To determine to what extent the primary amino acid sequence within exon 7 defines substrate specificity, exon 7 of FXIII was replaced with the corresponding exon of tTG using gene splicing by overlap extension. Other work from this laboratory (Achyuthan, K. E., Slaughter, T. F., Santiago, M. A., Enghild, J. J., and Greenberg, C. S. (1993) J. Biol. Chem. 268, 21284-21292) using synthetic peptides identified two other domains that might play a role in substrate recognition (located in exons 3 and 5). Therefore, recombinant chimeras of FXIII/tTG were also created in which these two exons were exchanged. FXIII, tTG, and chimeras 3, 5, and 7 were expressed in Escherichia coli, purified, and the nature of the fibrin cross-linking pattern of these five proteins was determined by immunoblot analysis. FXIII preferentially formed the gamma-gamma dimer, whereas tTG formed Aalpha-gamma complexes. Chimera 7 formed Aalpha-gamma complexes that resembled the cross-linking pattern of tTG. This finding demonstrates that the primary amino acid sequence of exon 7 of tTG confers some of the specificity for the Aalpha and Aalpha-gamma cross-link pattern characteristic of tTG. Chimera 5 exhibited reduced cross-linking activity (50% of FXIII activity) but still retained preference for formation of the gamma-gamma dimer, whereas chimera 3 was not active. In conclusion, exchanging the primary amino acid sequence of the active site exon of human FXIII with that of human tTG modifies the enzyme such that the fibrin cross-linking pattern more closely resembles that of tTG (Aalpha and Aalpha-gamma complexes) instead of FXIII (gamma-gamma dimers).