The effects of anabolic-androgenic steroid (AAS) abuse on the onset of puberty in female adolescents are largely unknown. This study assessed the acute effects of one AAS, stanozolol, on pubertal onset in the female rat. A single injection of stanozolol (5 mg/kg) on Postnatal Day (PN) 21 advanced vaginal opening but did not alter the onset of vaginal estrus. Higher doses of stanozolol treatment (10 and 25 mg/kg) also advanced vaginal opening but had no effect on vaginal estrus. The advancement of vaginal opening by stanozolol (5 mg/kg) was prevented by the concomitant administration of the pure antiestrogen ICI 182,780 (1 mg/kg) on PN20–22. Administration of the androgen receptor antagonist flutamide (10 mg/kg twice daily) on PN20–22 had no effect on the advancement of vaginal opening by stanozolol. Stanozolol treatment also advanced vaginal opening in ovariectomized rats. Perivaginal injections of a low dose of stanozolol (0.05 mg) on PN21 and PN23 also advanced vaginal opening. These results suggest that stanozolol is acting directly at estrogen receptors in the vaginal epithelium to advance vaginal opening and that prepubertal stanozolol treatment does not induce true precocious puberty.