Specific in vitro binding of [3H]testosterone (T), 5.alpha.-[3H]dihydrotestosterone (DHT) and [3H]estradiol (E2) was demonstrated in the 30,000 .times. g supernatant (cytosol) of thigh muscles (TM) and of the levator ani-bulbocavernosus muscle complex (LA-BC) by gel filtration through Sephadex G-25 columns. In TM cytosol, T and E2 are bound with high affinity (Ka = 1.1 .times. 109 M-1 and 2.3 .times. 109 M-1, respectively) whereas DHT binding is of lower affinity (Ka = 5.0 .times. 107 M-1). In LA-BC cytosol, T, E2, and DHT are bound with high affinity (Ka = 1.9 .times. 109 M-1, 0.3 .times. 109 M-1, and 0.5 .times. 109 M-1, respectively). Competition experiments suggest that the binding of the 3 hormones (T, E2 and DHT) is due to different proteins. In addition to TM and LA-BC, T and E2 binding was found in other muscles of male and female rats, including gastrocnemius, the pectoralis, diaphragm and heart.