Abstract The feasibility of performing steroid analysis by capillary gas chromatography on random urine samples for the detection of mild late-onset 21-hydroxylase deficiency was evaluated. Comparisons were made of basal excretions of androgen and 17α-hydroxyprogesterone metabolites with plasma levels (basal and stimulated) of 17α-hydroxyprogesterone and testosterone in six patients with the disorder. The following steroid metabolite excretion ratios were determined for normal controls and affected individuals. 1) 17α-hydroxypregnanolone /tetrahydrocortisone + tetrahydrocortisol + 5α-tetrahydrocortisol (cortisol metabolites) (normal 0.017 − 0.10, affected 0.17 − 0.42); 2) pregnanetriol/cortisol metabolites (normal 0.03 − 0.15, affected 0.17 − 0.99); 3) pregnanetriolone/cortisol metabolites (normal 0.02 − 0.014, affected 0.08 − 0.20); 4) androsterone + etiocholanolone/cortisol metabolites (normal 0.26 − 1.02, affected 0.34 − 1.47). Among the 21-deoxy steroid ratios, there was no overlap between affected and unaffected individuals. Two of six affected individuals had androsterone + etiocholanolone/cortisol metabolite ratios in the normal range. This method provides excellent discrimination between normal and affected individuals, precluding the need for an ACTH-stimulation test. It is anticipated that it will be increasingly used for diagnosis of the condition.