The juvenile hormone binding protein (JHBP) of larval Manduca sexta was labeled by a photoaffinity analog of JH II and purified by preparative IEF and ion-exchange HPLC. The purified [3H]EHDA-labeled JHBP was selectively cleaved by CNBr and by endoproteinases Lys-C and Glu-C. The radioactive peptides were separated by tricine SDS-PAGE and sequenced after blotting to a PVDF membrane. The sequence revealed that Ala184-Asn226 contained a primary binding site of [3H]EHDA. Furthermore, peptide mapping indicated that Asp1-Glu34 also contained a second covalent attachment site of [3H]EHDA. Labeling of the N-terminal region increased when the photolysis was performed at lower temperature. Since Ala184-Asn226 is predicted to be a hydrophobic beta-sheet region, it may participate in the recognition of lipophilic backbone of JH. Five out of six cysteines are located in these two regions, consistent with a model in which the two binding regions connected by disulfide bridges provide a two-sided binding pocket for juvenile hormone.