The SH3 domain from the Fyn tyrosine kinase possesses a buried hydrogen bond between the side chains of a glutamate (Glu24) and a serine (Ser41) residue. Multiple amino acid substitutions were made at these positions to determine the role of this interaction in the stability and conformational specificity of the domain and to assess the relationship between the thermodynamic stability of mutants and sequence conservation seen in the SH3 domain family. Analysis of single and double alanine mutations indicated that the Glu24-Ser41 interaction contributes 0.50 kcal/mol to the stability of the domain. However, disruption of the Glu24-Ser41 interaction did not impair peptide binding function, suggesting that the interaction is not critical for conformational specificity. The stability of the domain was not increased by the replacement of these residues with different combinations of hydrophobic residues or with potential salt bridge forming residues. Despite their similar structural roles in the Fyn SH3 domain, the Ser41 position was considerably more tolerant to substitution than was the Glu24 position. An alignment of >350 different SH3 domains has been completed in our laboratory. A statistically significant correlation was found between the conservation data for the Glu24 and Ser41 positions and the thermodynamic stabilities of the mutants constructed at these positions. Surprisingly, our analysis of sequence alignment data provided a more accurate prediction of the stability of mutants than did examination of the three-dimensional structure of the domain.