Women have higher circulating leptin levels than men. This sex difference is not simply explained by differences in the amount of body fat and is possibly influenced by their different sex steroid milieus. This prompted us to study prospectively the effects of cross-sex steroid hormones on serum leptin levels in 17 male to female transsexuals and 15 female to male transsexuals. Male to female transsexuals were treated with 100 micrograms ethinyl estradiol and 100 mg cyproterone acetate (antiandrogen) daily, and female to male transsexuals received testosterone esters (250 mg/2 weeks, im). Before and after 4 and 12 months of cross-sex hormone treatment, serum leptin levels and measures of body fatness were assessed. Before treatment, female subjects had higher serum leptin levels than male subjects independently of the amount of body fat (P < 0.01). Cross-sex hormone administration induced a reversal of the sex difference in serum leptin levels. Estrogen treatment in combination with antiandrogens in male subjects increased median serum leptin levels from 1.9 ng/mL before treatment to 4.8 ng/mL after 4 months and 5.5 ng/mL after 12 months of treatment (P < 0.0001). Testosterone administration in female subjects decreased median serum leptin levels from 5.6 to 2.6 ng/mL after 4 months and to 2.5 ng/mL after 12 months (P < 0.0001). Analysis of covariance revealed that the changes in serum leptin levels were independent of changes in body fatness in both groups (P < 0.01). In conclusion, these results indicate that sex steroid hormones, in particular testosterone, play an important role in the regulation of serum leptin levels. The prevailing sex steroid milieu, not genetic sex, is a significant determinant of the sex difference in serum leptin levels.