Studies were conducted to characterize Djungarian hamster androgen-binding protein and examine its expression during development. The cDNA encoding the full length testicular androgen-binding protein was cloned and, except for two suspected polymorphisms, shared a common primary sequence with hepatic sex hormone-binding globulin. A single androgen-binding protein/sex hormone-binding globulin gene was identified and a 1.7 kb mRNA encoding androgen-binding protein/sex hormone-binding globulin was present in both the testis and liver. Testicular homogenates contained specific 5 alpha-dihydrotestosterone-binding activity that was identified as androgen-binding protein. In the prepubertal testis, immunoreactive androgen-binding protein/sex hormone-binding globulin subunits ranged from 46 kDa to 60 kDa, with the majority of isoforms > 50 kDa. These subunits were distinct from the major 55 kDa and 51 kDa isoforms of sex hormone-binding globulin present in the serum of prepubertal hamsters. Deglycosylation studies demonstrated that size heterogeneities were the result of developmentally specific glycosylation patterns. Expression of androgen-binding protein by the testis was upregulated during puberty and coincided with a decline in serum sex hormone-binding globulin activity. The tissue- and age-dependent expression of specific androgen-binding protein and sex hormone-binding globulin variants suggests that these proteins play different roles in steroid-mediated sexual development.