Human sex steroid-binding protein (hSBP/hABP or hSHBG) was over-expressed in High Five and Sf9 cells adhered to plates and in suspension. The adherent cells expressed to levels of 2.3 mg/l and 1.4 mg/l after 4 and 6 days, respectively, while Sf9 cells grown in suspension yielded 4.67 mg/l after 6 days. Recombinant hSBP/hABP, purified to homogeneity by immunoadsorption, was found to fold similarly to native plasma hSBP/hABP and to display similar sequence epitopes after heat denaturation. The recombinant protein binds dihydrotestosterone, testosterone, and 17 beta-estradiol with KdS of 0.6, 2.4, and 14.2 nM, respectively, which are similar to plasma hSBP/hABP. The recombinant protein contains N-linked and O-linked oligosaccharide side-chains but the monomer exhibits a slightly lower molecular weight than plasma hSBP/hABP (40 kDa vs 44 kDa) which may be due to the absence of one N-linked side-chain or to shorter oligosaccharide side-chains. The partial N-terminal sequence LRPVLP(T)Q of recombinant hSBP/hABP is identical to plasma hSBP/hABP but appears to be less heterogeneous. These results indicate that recombinant baculovirus SBP represents a good model for investigating the structure of plasma hSBP/hABP. The expression system will allow the isolation of preparative amounts of SBP mutants generated by combinatorial site-directed mutagenesis to advance investigations on structure-function relationships and undertake crystallization trials for X-ray diffraction analyses.