Studies on the glutamine substrate specificities of human plasma factor XIIIa and guinea pig liver transglutaminase have been made using variants of the synthetic peptide substrate, Ser-Val-Leu-Ser-Leu-Ser-Gln-Ser-Lys-Val-Leu-Pro-Val-Pro-Glu. The sequence of this effective peptide substrate corresponds to the primary site of factor XIIIa-catalyzed amine incorporation into beta-casein, the most sensitive known macromolecular substrate for this enzyme (Gorman, J.J., and Folk, J.E. (1980) J. Biol. Chem. 255, 419-427). Variations in specificity observed with factor XIIIa for peptides containing single substitutions and multiple substitutions in this sequence are indications that several important determinants for enzyme recognition are contained therein. Among these are several of the hydrophobic amino acid residues and the lysine residue. Less pronounced changes in specificity occur with the liver enzyme and the differences in effects of the various substitutions reveal important differences in specificity requirements of factor XIIIa and the liver enzyme. Comparisons of the activities of the enzymes toward the synthetic peptides to their activities toward macromolecular substrates suggest that higher order macromolecular structural features contribute to specificity.