The structure of Escherichia coli-derived rat intestinal fatty acid- binding protein (I-FABP) exhibits a β-clam topology comprised of two five- stranded antiparallel β-sheets surrounding a large solvent-filled cavity into which the ligand binds. It also contains two α-helices that span residues E15-A32 and join β-strands A and B. This helical domain is conserved in all proteins of this family for which structures have been determined. In order to assess the structural and functional role of the helical domain, we engineered a variant of I-FABP by deleting residues 15-31 and inserting a Ser-Gly linker after residue 14. Circular dichroism measurements indicated that this I-FABP variant, termed Δ17-SG, has a high β-sheet content similar to that of the wild-type protein. Two-dimensional NMR spectra of Δ17-SG revealed patterns similar to those observed for wild- type I-FABP, except for the selective absence of resonances and through- space interactions assigned to the helical domain. The Δ17-SG variant was less stable to denaturant than wild-type I-FABP, but the folding-unfolding transition was highly cooperative and reversible. Taking into account the lower stability, the refolding kinetics of Δ17-SG were essentially identical to those of wild-type. We conclude that Δ17-SG is a helix-less, essentially all-β-sheet variant of I-FABP and that the helical domain is not a required element of the β-clam topology of I-FABP. In addition, the helical domain does not appear to serve as a nucleation site for the refolding process. As shown in the accompanying paper [Cistola, D. P., Kim, K., Rogl, H., and Frieden, C. (1996) Biochemistry 35, 7559-7565], the helices may function to regulate the kinetics and energetics of ligand binding.