In vitro testosterone binding was studied in a nuclear extract (0.15 M KC1) from rat bone marrow using the charcoal assay. The nuclear extract contains binding sites saturable at low concentration of testosterone (1 times 10-8M). From a Scatchard plot of the data obtained it is concluded that a single class of receptor sites is involved in the binding of testosterone. Studies indicated that the testosterone-nuclear complex has a dissociation constant (Kd) of 5.9 times 10-9 M binding 9 times 10-14 moles/mg nuclear protein. 5alpha- and 5beta-dihydrotestosterone compete poorly with testosterone for binding sites in the nuclear component while estradiol -17beta does not compete at all. The protein nature of the receptor was demonstrated and an isoelectric point (pI) of 4.9 was determined for the complex by the use of isoelectric focusing.