We have used site-directed mutagenesis to investigate the contributions of widely distributed residues in the thymosin beta-4 (Tbeta4) sequence to the formation and stability of the actin-Tbeta4 complex. Equilibrium and kinetic studies of actin binding were performed by monitoring the change in fluorescence of an N-iodoacetyl-N9-(5-sulfo-1-naphthyl)ethylenediamine (Aedans) fluorophore on actin cysteine-374. We evaluated the contributions of hydrophobic residues throughout Tbeta4, the conserved and variable proline residues, and the conserved lysine residues to the kinetics and thermodynamics of Tbeta4 binding to MgATP-actin monomers. Pro4, Lys18, Lys19, Pro27, Leu28, Pro29, and Ile34 were substituted by alanine residues. All these mutations weaken the affinity of the actin-Tbeta4 complex, but the kinetic basis of the lower stability of the complex varies among the mutants. Our results support a model in which Tbeta4 initially binds actin through an electrostatic interaction, followed by the formation of widely distributed hydrophobic contacts. Several mutants, particularly at proline residues, dissociate much more rapidly than the wild-type complex, but also show small increases in the rate of association. This seeming paradox suggests that conformational searching of Tbeta4, and particularly cis-trans isomerization of proline residues, contributes to the slow association rate constant of Tbeta4, and to the stability of the hydrophobic contacts associated with strong actin binding.