Complexes between Src-homology 3 domains and proline-rich target peptides can have lifetimes on the order of milliseconds, making them too short-lived for kinetic characterization by conventional methods. Nuclear magnetic resonance (NMR) dynamics experiments are ideally suited to study such rapid binding equilibria, and additionally provide information on partly bound intermediate states. We used NMR together with isothermal titration calorimetry (ITC) to characterize the interaction of the SH3 domain from the Fyn tyrosine kinase with a 12-residue peptide at temperatures between 10 and 50 degrees C. NMR data at all temperatures are consistent with an effectively two-state binding reaction, such that any intermediates are either very weakly populated or exchange extremely rapidly with the free or bound forms. Dissociation rate constants, determined by CPMG and ZZ-exchange NMR experiments, range from k(off)(10 degrees C) = 4.5 s(-1) to k(off)(50 degrees C) = 331 s(-1). ITC data at all temperatures follow a simple two-state interaction model. Binding is favored enthalpically, with a dissociation enthalpy, Delta H-D(30 degrees C) = 15.4 kcal mol(-1), and disfavored entropically, with a dissociation entropy, Delta S-D(3 degrees C) = 20.0 cal mol(-1) K-1. The free protein and peptide have significantly higher heat capacity than the bound complex, Delta C-p = 352 Cal mol(-1) K-1, which is consistent with the largely hydrophobic character of the binding interface. An Eyring plot of k(off) values gives an activation enthalpy of dissociation, Delta H-D(double dagger)(30 degrees C) = 19.3 kcal mol(-1) and exhibits slight curvature consistent with the ITC-derived value of Delta C-p. The curvature suggests that nonpolar residues of the hydrophobic interface are solvated in the transition state for dissociation. Association rate constants were calculated as k(on) = k(off)/K-D, and range from k(on)(10 degrees C) 1.03 x 10(8) M-1 s(-1) to k(on)(50 degrees C) = 2.0 x 10(8) M-1 s(-1), with an apparent activation enthalpy, Delta H-A(double dagger) 3.4 kcal mol(-1). Both the magnitudes and temperature dependence of k(on) values are consistent with a diffusion-limited association mechanism. The combination of NMR and ITC data sheds light on how the Fyn tyrosine kinase is activated by binding to proline-rich targets, and represents a powerful approach for characterizing transient protein/ligand interactions.