Urinary steroid excretion after androstenedione intake has been examined after a single dose of 50 mg and single doses of 100 or 300 mg/d for 7 d. We evaluated the effects of 28 d of 100 mg three times a day (t.i.d.) androstenedione intake on urinary steroid excretion. Twenty healthy men, ages 30-39 yr (33.5 +/- 0.6), consumed 100 mg androstenedione t.i.d. or placebo for 28 d. Urine samples were analyzed for testosterone, epitestosterone, androsterone, and etiocholanolone via HPLC/tandem mass spectrometry on d 0 and 28. Androstenedione intake increased (P < 0.05) urinary testosterone 35.1 +/- 10.5 ng/ml vs. 251.6 +/- 87.5 ng/ml, epitestosterone 35.3 +/- 8.8 ng/ml vs. 99.7 +/- 28.7 ng/ml, androsterone 2,102 +/- 383 ng/ml vs. 15,767 +/- 3,358 ng/ml, and etiocholanolone 1,698 +/- 409 ng/ml vs. 11,329 +/- 2,656 ng/ml (means +/- se). Although the testosterone to epitestosterone ratio (T/E) tended to increase with androstenedione intake (1.2 +/- 0.3 vs. 4.0 +/- 1.6; P = 0.12), only one subject had a urinary T/E greater than the current Olympic criteria (>6.0) for a positive drug test. Chronic intake of 100 mg androstenedione t.i.d. increases the urinary excretion of steroid metabolites. Due to inconsistent increases in the T/E ratio, the T/E ratio may not effectively detect androstenedione use.