Photolabeled testosterone-binding globulin (TeBG), obtained from the plasma of sexually immature male rabbits, was produced using 17β-hydroxy-[l,2-3H]4,6-androstadien-3-one. Photolabeled TeBG could not be distinguished from unlabeled TeBG by gel filtration chromatography, electrophoresis on nondenaturing gels, or sucrose gradient ultracentrifugation. Rabbit TeBG had a Stokes radius of approximately 45 Å and a sedimentation coefficient of approximately 4.6S. Based on these parameters, its native molecular weight was calculated to be approximately 78,000. The frictional ratio of rabbit TeBG was found to be approximately 1.6. When photolabeled TeBG was examined on polyacrylamide gels containing sodium dodecyl sulfate, a single androgen-specific peak of approximately 40,000 daltons was obtained. When photolabeled TeBG was treated with the cross-linking reagent disuccinimidyl suberate before electrophoresis on sodium dodecyl sulfate gels, an additional androgen-specific peak of radioactivity corresponding to approximately 100,000 daltons was obtained. We interpret this peak to represent oligomers of the TeBG subunits. The 40,000-dalton subunit was obtained regardless of whether the proteins were treated with 2-mercaptoethanol, indicating that the monomers are not linked by disulfide bonds.