We studied the possible existence of physiological sex differences in serum aminopeptidase activities in mice, by evaluating the effect of gonadectomy and the in vitro response to the presence in the medium of cholesterol or steroid hormones. Alanyl- and glutamyl-aminopeptidase activities were measured in sera from male, female, orchiectomized and ovariectomized mice, incubated with substrate solutions, and compared with the same groups of serum incubated with substrate solutions including cholesterol, 17-beta-estradiol, testosterone, progesterone or hydrocortisone. Our results demonstrated highly significant sex differences, and an influence of cholesterol and steroid hormones on aminopeptidase activity. Depending on the nature of the aminopeptidase, these enzymes responded in different ways to the presence of these substances and also responded differently to gonadectomy. For alanyl-aminopeptidase activity, but not for glutamyl-aminopeptidase activity, there was a clear difference in response between males and females to incubation of the serum with steroid hormones.