A cDNA for the hemolymph juvenile hormone binding protein (JHBP) of larval Manduca sexta has been isolated, sequenced, and expressed in an insect cell line. A recombinant baculovirus, containing the JHBP cDNA fused to the p10 promoter of Autographa californica nuclear polyhedrosis virus, was constructed. Insect cells (Sf9) infected with this virus secreted recombinant JHBP (rJHBP) into the medium (> 50 micrograms/mL), and cotranslational removal of an 18 amino acid leader sequence was observed. rJHBP was cross-reactive with an antiserum prepared to the hemolymph JHBP and was specifically labeled by [3H]EHDA, a photoaffinity analog of JH II, demonstrating that rJHBP was an isoform of the previously reported 32-kDa JHBP [Lerro, K. A., & Prestwich, G.D. (1990) J. Biol. Chem. 265, 19800-19806]. rJHBP was purified from insect cell medium to homogeneity by ion-exchange and gel-filtration chromatography. The purified rJHBP had a higher affinity (KD = 11 nM for JH I and KD = 42 nM for JH II) than that reported for crude hemolymph JHBP (KD = 80 nM for JH I). The circular dichroism (CD) spectrum of purified rJHBP indicated 34% alpha-helix and 23% beta-sheet. The CD spectra of rJHBP in the presence and absence of JH II were the same, indicating no change in secondary structure induced by ligand binding. Thus, the rJHBP expressed in insect cells binds JHs and is suitable for structural and functional analysis.