Intestinal fatty acid-binding protein (I-FABP) binds a single molecule of long-chain fatty acid in an enclosed cavity surrounded by two antiparallel β-sheets. The structure also contains two short α-helices which form a cap over one end of the binding cavity adjacent to the methyl terminus of the fatty acid. In this study, we employed a helix-less variant of I-FABP known as Δ17-SG [Kim, K., Cistola, D. P., and Frieden, C. (1996) Biochemistry 35, 7553-7558] to investigate the role of the helical region in maintaining the integrity of the binding cavity and mediating the acquisition of ligand. Fluorescence and NMR experiments were used to characterize the energetic, structural, and kinetic properties of fatty acid binding to this variant, and the results were compared and contrasted with those of wild-type I-FABP and a single-site mutant, R106T. Remarkably, oleate bound to Δ17-SG with a dissociation constant of 4.5 μM, a value comparable to that for R106T and approximately 20-100-fold higher than that for wild-type I-FABP. Heteronuclear two-dimensional NMR spectra for [2-13C]palmitate complexed with Δ17-SG revealed a pattern nearly identical to that observed for the wild-type protein, but distinct from that for R106T. In addition, the ionization behavior of bound [1-13C]palmitate and the nearest neighbor patterns for [2-13C]palmitate derived from 13C-filtered NOESY experiments were very similar for Δ17-SG and the wild-type protein. These results implied that the fatty acid-protein interactions characteristic of the carboxyl end of the fatty acid binding cavity in the wild-type protein were essentially intact in the helix-less variant. In contrast, 13C-filtered NOESY spectra of [16-13C]palmitate bound to Δ17-SG indicated that the fatty acid-protein interactions at the methyl end of the binding cavity were disrupted. As determined by stopped-flow fluorescence, the observed ligand association rates for both Δ17-SG and wild-type I-FABP increased with increasing oleate concentration, but only the wild-type protein exhibited a limiting value of 1000 s-1. This rate-limiting process was interpreted as a conformational change involving the helical region that allows the ligand access to the internal cavity. Simulation and fitting of the kinetic results yielded ligand association rates for Δ17-SG and wild-type I-FABP that were comparable. However, the dissociation rate for wild-type protein was 16-fold lower than that for Δ17-SG. We conclude that the α-helices of I-FABP are not required to maintain the integrity of the fatty acid binding cavity but may serve to regulate the affinity of fatty acid binding by selectively altering the dissociation rate constant. In this manner, conformational changes involving the α-helical domain may help control the transfer of fatty acids within the cell.