The ligand specificities and binding affinities of three recombinant pheromone binding proteins (PBPs) of two saturniid moths (genus Antheraea) were determined by using a novel binding assay in conjunction with two tritium-labeled constituents of the pheromone blend, [3H]-6E,11Z-hexadecadienyl acetate and [3H]-4E,9Z-tetradecadienyl acetate. The new binding assay, in which nonspecific adsorption to a plastic vessel is suppressed by presaturation of the surface with a 1-alkanol, allows measurement of dissociation constants (KD) for lipophilic ligands for their carrier proteins. The three PBPs showed KD values for [3H]-6E,11Z-16:Ac and [3H]-4E,9Z-14:Ac between 0.6 and 30 microM, as determined by Scatchard analysis. Importantly, two PBPs (Aper-1 and Aper-2) from one species showed opposite binding specificities for these two ligands. Aper-1, like Apol-3, showed 15-fold higher affinity for 6E,11Z-16:Ac than for 4E,9Z-14:Ac, while Aper-2 showed a 3.5-fold preference for binding the shorter chain compound. In addition, for the Apol-3 PBP, displacement of [3H]-6E,11Z-16:Ac binding by other pheromone components or analogs showed a clear trend in relative binding affinity: 6E,11Z-16:Ac > 4E,9Z-14:Ac > 6E,11Z-16:Al approximately 16:Ac > 6E,11Z-16:OH > 4E,9Z-14:OH. These data clearly demonstrate a > 1000-fold range of binding affinities among these very similar structures and unambiguously demonstrate the specificity of the PBP-pheromone interaction. Moreover, this assay offers the potential for determining ligand specificities for odorant binding proteins and other proteins in the vertebrate lipocalin superfamily.