Pheromone perception in Lepidoptera requires initial recognition and transport of the pheromone molecule by ligand-specific pheromone binding proteins (PBPs) in the moth antennae, followed by recognition of the ligand or PBP-ligand complex by a transmembrane G-protein-coupled odorant receptor protein. This signal is transduced by activation of a specific phospholipase C, intracellular release of inositol 1,4,5-trisphosphate (IP3) and IP3-gated opening of an ion channel. Individual pheromone-specific PBPs provide the initial ligand recognition event and encode ligand specificity. We have used photoaffinity labeling, cDNA library screening and cloning, protein expression, a novel binding assay and site-directed mutagenesis to define the ligand specificity of PBPs.