A double isotope ratio technique was used to estimate the specific binding of testosterone (T), as opposed to its biologically nonactive stereoisomer, epitestosterone (Epi T). The mouse erythropoietic spleen formed in response to a phenylhydrazine-induced hemolytic anemia was used as the target organ. Spleen minces from preanemic mice, as well as those in the early and late phases of erythropoietic spleen development, were incubated with 10(-9) M of 14C-T and 3H-EpiT, and the selective uptake of T was calculated from the 14C/3H ratio measured in the media before and after incubation, as well as in the subcellular fractions of the minces. Preferential uptake of T was seen in the early phase of development, but not in spleens obtained from preanemic animals or those in the late phase. There was no evidence of metabolic conversion of T or EpiT. The selective uptake of T by early phase spleens was reflected in a preferential nuclear accumulation of T. These data represent the first demonstration of a specific binding of T in vitro to a developing erythroid tissue.