An abundant cytosolic protein from hamster Harderian glands was partially purified by ultracentrifugation and analyzed on SDS-PAGE. This 156-kDa protein occurs in male but not in female glands. Castration of adult males for different time periods resulted in a gradual disappearance of the protein over 5 weeks. Daily administration of testosterone, 5 alpha-dihydrotestosterone, 3 alpha- or 3 beta-androstanediol (0.5 mg/day/15 days), to castrated males maintained the levels of the protein. In females, the protein was induced with the same compounds but not with 17 beta-estradiol. These observations indicate an androgen-mediated hormonal regulation. In developing animals, the 156-kDa protein was detectable in glands of 2-week-old males and females; it was not detected in 4-week-old females. Its specific reactions to 3,3',5,5'-benzidine derivatives strongly suggest heme groups associated with this Harderian gland protein.