Factor XIIIa (FXIIIa) stabilizes fibrin clots by covalently cross-linking fibrin molecules. The purpose of this study was to determine the amino acid requirements at the active site of FXIIIa for catalysis. We selected amino acids 310-317 (Arg-Tyr-Gly-Gln-Cys-Trp-Val-Phe) in the human FXIII A-chain sequence for analysis based on the high degree of sequence homology among the different transglutaminases. We converted each amino acid in this region to Ala by site-directed mutagenesis. These recombinant FXIII A-chain mutants were expressed in Escherichia coli using the pTrc99A expression vector. FXIIIa activity was assessed by measuring the incorporation of 5-(biotinamido)pentylamine into N,N'-dimethylcasein in a solid-phase microtiter plate assay. The Cys-314-->Ala mutation yielded a recombinant protein with no FXIIIa activity. We also found that changing Gly-312 and Val-316 to Ala resulted in 22 and 65% decreases in activity, respectively. The other five mutations near the active-site Cys resulted in FXIIIa molecules in which the activity was reduced > 95%. The mechanism of SH protease catalysis is similar to transglutaminase catalysis in that both form thioester intermediates. His and Asp residues may stabilize this enzyme-substrate intermediate. Therefore, we performed site-directed mutagenesis on several His residues (His-342, His-373, and His-450) as well as Asp-396 in human FXIII. We found that changing His-342 to Ala reduced catalytic activity by 85%, while the His-373-->Ala mutant had no activity. In contrast, changing His-450 to Ala reduced FXIIIa activity by only 15%. We also examined the activity of all the mutants in a fibrin cross-linking assay. Four of the mutations (Phe-317-->Ala, Tyr-315-->Ala, Gln-313-->Ala, and Asp-396-->Ala), in which the activity toward the small primary amine was reduced by > 95%, were still capable of cross-linking the gamma-chain of fibrin. Even though these four mutants produced gamma-gamma dimers, they were not capable of forming higher molecular weight cross-linked products. Finally, we found that the binding of all the mutants to fibrin was similar to that of wild-type FXIIIa. In conclusion, we demonstrated that changing the specific amino acids Arg-310-Phe-317 to Ala substantially reduced FXIIIa activity. In addition, full catalytic activity was dependent on His-342, His-373, and Asp-396. These findings provide new insights into the catalytic mechanism of FXIIIa.