In insects, hydrophobic odorants are transported through the sensillar lymph to receptors on sensory neurons by odorant-binding proteins (OBPs). The beetle, Tenebrio molitor, which is a pest of stored grain products, produces a set of 12-14 kDa OBP-like proteins in their hemolymph. The structure of one of these proteins and that of a moth pheromone-binding protein have been solved. Both proteins have at least six -helices with an internal, hydrophobic, ligand-binding pocket, but the beetle OBP lacks one of the disulfide bonds immediately adjacent to this pocket. To explore this difference and to sample isoform diversity, T. molitor hemolymph OBPs were fractionated by gel-exclusion chromatography and reversed-phase HPLC. Selected fractions were reduced and alkylated, and tryptic peptides were sequenced by MS/MS. Partial sequences of 7 different isoforms were obtained and used to clone 9 new cDNAs encoding OBPs with identities from 32 to 99%. The more divergent isoforms have numerous substitutions of hydrophobic residues that presumably alter the shape and specificity of the ligand-binding pocket. These isoforms all lack the same third disulfide bridge and are more similar to one another than to any of the 38 OBPs in Drosophila melanogaster. They have presumably arisen via gene duplication following separation of the major insect orders.