An analysis of the data collected in the Quebec Overfeeding Study of identical twins was undertaken to determine any evidence of a genotype effect on plasma levels of adrenal and gonadal steroids arising from long term positive energy balance. Plasma levels of sex hormone-binding globulin (SHBG), testosterone, dihydrotestosterone (DHT), dehydroepiandrosterone sulfate (DHEA-S), androsterone glucuronide, androstane-3 alpha, 17 beta-diol glucuronide (3 alpha-DIOL-G), and cortisol were measured in 12 pairs of young, sedentary, male monozygotic twins before and after 100 days of overfeeding. The dietary energy excess of 4.2 MJ/day (1000 Cal), 6 days a week, resulted in a total positive energy balance of 353 MJ (84,000 Cal). Overfeeding induced significant changes (P < 0.0001) in body weight and other measures of body composition. Within-twin pair resemblance was observed at baseline in all steroids, except cortisol [intraclass correlation range: DHEA-S, 0.50 (P < 0.05); DHT, 0.77 (P < 0.001)] and was lost with overfeeding, except for DHT and SHBG (P < 0.05). SHBG levels fell and 3 alpha-DIOL-G rose with the gain in body fatness. The change in testosterone was a significant correlate of the change in upper body fat (r = -0.48; P < 0.05). The change in 3 alpha-DIOL-G correlated positively with increases in all measures of central adiposity (r = 0.52; P < 0.01). A decrease in DHEA-S occurred with a higher, but not with a lower, gain in abdominal visceral fat (P < 0.05). Thus, analysis of adrenal and gonadal steroids and of conjugated metabolites before and after overfeeding in monozygous twins supports the idea that there is a genotype effect on steroid circulating steroid levels and that these blood levels are correlated with the pattern of body fat distribution. Moreover, the baseline within-twin pairs similarity in steroid levels was attenuated by prolonged positive energy balance and body fat gain.