The baculovirus system is able to generate large amounts of a protein, permitting detailed analysis of structure-function relations. We have used this system to overexpress and characterize normal human androgen receptor (hAR) and mutant hARs from humans with complete or partial androgen insensitivity. Maximum specific binding of [3H]mibolerone (MB) in recombinant baculovirus-infected Spodoptera frugiperda (Sf9) cells varied from 15 to 40 pmol/mg protein, about 1000-fold higher than in genital skin fibroblasts, and peaked 48–72 h after infection. In contrast, Coomassie blue staining and Western blotting revealed maximum accumulation of 110–120 kDa hAR proteins 96 h post-infection. Normal and mutant hARs were specifically photoaffinity-labeled with [3H]methyltrienolone (MT), and had normal steroid-binding selectivity: the order of competition was androgen>estrogen>progestin>glucocorticoid. Normal hAR was phosphorylated in Sf9 cells, reacted with antibodies against phosphoserine and phosphothreonine after purification using testosterone-biotin, and transactivated a transfected androgen response element-luciferase reporter in infected Sf9 cells. Two mutant hARs had increased rates of dissociation from MB and MT that were in accord with the associated degree of clinical androgen insensitivity: complete, Pro903Ser>partial, Leu820Val; the third, Ile663Asn, was not abnormal. Our data extend the characterization of normal hAR produced by baculovirus-infected Sf9 cells, and demonstrate, for the first time, that point-mutated hARs so produced can display distinctive biochemical phenotypes.