Testosterone replacement in hypogonadism has long been known to promote nitrogen retention and increase body density, but the mechanisms of nitrogen retention and body composition changes are poorly defined. We measured body composition and muscle protein synthesis in five hypogonadal men before and 6 months after initiating testosterone replacement. Body composition was examined using dual energy X-ray absorptiometry. Muscle mass was estimated both by excretion of creatinine on a meat-free diet and from appendicular mass measured using dual energy X-ray absorptiometry. Muscle protein synthesis was assessed by measuring the increment of [13C]leucine in mixed muscle protein and myosin heavy chain during a continuous infusion of L-[l-13C]leucine. In all subjects there was an increase in fat-free mass (average, 15%; range, 10-22%; P = 0.02) and a decrease in fat mass (-11%; range, -0.4% to -22.0%; P = 0.03). Muscle mass also increased in everybody (mean, 20%; range, 11-32%; P = 0.04) such that 65% of the increase in fat-free mass could be attributed to accretion of muscle. The accumulation of muscle was associated with a 56% (P = 0.015) increase in the fractional synthesis rate of mixed skeletal muscle proteins and a trend toward a similar increase in the fractional synthesis rate of myosin heavy chain (46%; P = 0.098). We conclude that testosterone replacement in hypogonadal men enhanced skeletal muscle mass by stimulating the muscle protein synthesis rate.