The thermodynamics of binding of unfolded polypeptides to the chaperone SecB was investigated in vitro by isothermal titration calorimetry and fluorescence spectroscopy. The substrates were reduced and carboxamidomethylated forms of RNase A, BPTI, and $α$-lactalbumin. SecB binds both fully unfolded RNase A and BPTI as well as compact, partially folded disulfide intermediates of $α$-lactalbumin, which have 40-60% of native secondary structure. The heat capacity changes observed on binding the reduced and carboxamidomethylated forms of $α$-lactalbumin, BPTI, and RNase A were found to be -0.10, -0.29, and -0.41 kcal $mol^{-1} K^{-1}$, respectively, and suggest that between 7 and 29 residues are buried upon substrate binding to SecB. In all cases, binding occurs with a stoichiometry of one polypeptide chain per monomer of SecB. There is no evidence for two separate types of binding sites for positively charged and hydrophobic ligands. Spectroscopic and proteolysis protection studies of the binding of SecB to poly-L-Lys show that binding of highly positively charged peptide ligands to negatively charged SecB leads to charge neutralization and subsequent aggregation of SecB. The data are consistent with a model where SecB binds substrate molecules at an exposed hydrophobic cleft. SecB aggregation in the absence of substrate is prevented by electrostatic repulsion between negatively charged SecB tetramers.