Exposure to novel adult males and their urine can hasten the onset of sexual maturity in female mice. Some evidence implicates chemosignals from males’ preputial glands, while other evidence suggests that male urinary steroids, especially 17β-oestradiol, contribute to this effect. The present experiment was designed to determine whether preputial gland removal would influence the capacity of males to accelerate female sexual development, and to measure male urinary oestradiol and testosterone in the presence or absence of these glands. Juvenile females aged 28 days were housed for two weeks in isolation or underneath two outbred males that had undergone preputialectomy or sham surgery. Urine samples were collected non-invasively from males that were isolated or exposed to females, then assayed for oestradiol, testosterone and creatinine. Combined uterine and ovarian mass from females sacrificed at 43 days of age was increased by exposure to males, regardless of whether or not these males had been preputialectomised. Male urinary creatinine was reduced by exposure to developing females. Creatinine-adjusted oestradiol and testosterone were significantly greater in female-exposed than in isolated males, in both preputialectomised and intact males. These data suggest that the preputials are not necessary for the capacity of males to hasten female uterine and ovarian growth. As exogenous oestrogens can promote uterine growth and other parameters of female reproductive maturation, oestradiol in males’ urine may contribute to earlier sexual maturity in male-exposed females.