Circulating testosterone is elevated acutely following resistance exercise (RE) and is an important anabolic hormone for muscle adaptations to resistance training. The purpose of this study was to examine the acute effect of heavy RE on intracrine muscle testosterone production in young resistance-trained men and women. Fifteen young, highly resistance-trained men (n = 8; 21 +/- 1 yr, 175.3 +/- 6.7 cm, 90.8 +/- 11.6 kg) and women (n = 7; 24 +/- 5 yr, 164.6 +/- 6.7 cm, 76.4 +/- 15.6 kg) completed 6 sets of 10 repetitions of Smith machine squats with 80% of their 1-repetition maximum. Before RE and 10 and 70 min after RE, muscle biopsies were obtained from the vastus lateralis. Before RE, after 3 and 6 sets of squats, and 5, 15, 30, and 70 min into recovery from RE, blood samples were obtained using venipuncture from an antecubital vein. Muscle samples were analyzed for testosterone, 17beta-hydroxysteroid dehydrogenase (HSD) type 3, and 3beta-HSD type 1 and 2 content. Blood samples were analyzed for glucose and lactate concentrations. No changes were found for muscle testosterone, 3beta-HSD type 1 and 2, and 17beta-HSD type 3 concentrations. However, a change in protein migration in the Bis-Tris gel was observed for 17beta-HSD type 3 postexercise; this change in migration indicated an approximately 2.8 kDa increase in molecular mass. These findings indicate that species differences in muscle testosterone production may exist between rats and humans. In humans, muscle testosterone concentrations do not appear to be affected by RE. This study expands on the current knowledge obtained from animal studies by examining resting and postexercise concentrations of muscle testosterone and steroidogenic enzymes in humans.