We have designed a model protein that consists of two identical 35-residue polypeptide chains, parallel and in-register arranged in a two-stranded alpha-helical coiled-coil structure. This structure is stabilized by interchain hydrophobic interactions between leucine residues at positions "a" and "d" of a repeating heptad sequence. To determine the positional effects of interchain hydrophobic interactions on the stability of the coiled-coil, a single leucine residue in each chain at position "a" (9, 16, 23, 30) and "d" (5, 12, 19, 26, 33) was systematically replaced by an alanine. All these proteins formed two-stranded alpha-helical coiled-coils in benign conditions (0.05 M phosphate, 0.1 M KCl, pH 7). The stability of each mutant protein was determined by guanidine hydrochloride denaturation experiments, where the decrease in ellipticity at 220 nm was monitored by circular dichroism. The single alanine replacements of a leucine residue at hydrophobic positions a and/or d are all shown to destabilize the coiled-coil structure. The non-equivalent hydrophobic positions a and d make an equivalent contribution to protein stability along the majority of the coiled-coil structure (positions 9-30). The small decrease in coiled-coil stability caused by Leu----Ala substitution at either ends of the coiled-coil suggested that the Leu-Leu hydrophobic interactions are less important at the ends of the coiled-coil and the ends of the coiled-coil are more flexible. Analysis of the difference between the ellipticity in benign buffer and in 50% trifluoroethanol (delta theta 220) and the slope term from a plot of the free energy of unfolding versus guanidine hydrochloride concentration also supported the conclusion that the leucine residues at the ends of the coiled-coil are much less buried than in the middle section of the coiled-coil.