Src homology 2 (SH2) domains are protein modules that specifically bind to tyrosyl phosphorylated peptides on signaling proteins. X-ray crystallographic studies of the SH2 domain of the Src kinase have probed the mechanism of binding, leading to the "two-pronged plug two-holed socket" mechanism whereby binding is hypothesized to resemble a two-pronged plug (the peptide) inserting into a two-holed socket (the SH2 domain). This binding model predicts (1) a hydrophobic basis for high-affinity binding largely determined by the level of insertion of the third residue C-terminal to the phosphotyrosine in the peptide into a primarily hydrophobic pocket (the +3 binding pocket) of the SH2 domain, and (2) a binding mechanism involving no significant conformational changes in the SH2 domain. In this study, we have probed these predictions by using isothermal titration calorimetry to extract complete thermodynamic profiles (Delta G degrees, Delta H degrees, Delta S degrees, Delta Cp degrees) for the binding of the Src SH2 domain to two series of tyrosyl phosphopeptides. One series consisted of peptides that have been determined by X-ray crystallography to have different levels of insertion of the peptide's +3 position into the +3 binding pocket. The other series consisted of peptides with progressively smaller hydrophobic side chains (I, L, V, and A) at the +3 position. Consistent with a binding mechanism that does not involve substantial conformational changes, the Delta Cp degrees values for all peptides were small and, at least for the high-affinity interactions, similar to the Delta Cp degrees values predicted from surface area calculations. However, unexpectedly, this study reveals that high-affinity binding was only partially determined by the interactions between the +3 residue in the peptide and the +3 binding pocket. Furthermore, the Delta Cp degrees values for all peptides studied were similar, implying similar degrees of desolvation of the +3 binding pocket upon binding. These results indicate that the "two-pronged plug two-holed socket" model is an oversimplification of the Src SH2 domain binding mechanism.