<jats:p> Animal studies show significant differences in steroid metabolism between male and female subjects. Similar studies in human subjects are still needed. The aim of this study was to evaluate differences in 24-h urinary excretion of cortisol and androgen metabolites between healthy male and female volunteers to estimate if such differences were significant. Urinary metabolite measurements were performed by gas chromatography. The median urinary excretion of total cortisol metabolites was 6965 μg/day for men and 4595 μg/day for women ( P=0·0005). Urinary excretion of 11 β-hydroxyandrosterone, tetrahydrocortisone, tetrahydrocortisol (5 β), allotetrahydrocortisol (5 α), α-cortolone, β-cortolone + β-cortol and α-cortol were also significantly different in men compared with women. Total androgen metabolites in men (2660 μg/day) were also higher than in women (1850 μg/day) ( P< 0·0003). Similarly, urinary excretion of androsterone (5 α), aetiocholanolone (5 β) and dehydroepiandrosterone were also significantly greater (all P=0·01). This confirms significant differences in the steroid metabolite excretion profiles between men and women. Laboratories should consider adopting gender-related reference ranges for cortisol and androgen metabolite excretion in 24-h urine samples.