The uptake of androgens into the nuclei of caput epididymis, ventral prostate, seminal vesicle and testis was studied by recirculating physiological and pharmacological concentrations of [3H]testosterone in an artificial medium through the lower half (hemicorpus) of castrated or hypophysectomized rats. The accumulation of dihydrotestosterone in accessory sex organ nuclei was saturable, inhibited by perfusion of excess testosterone or cyproterone acetate, and associated with binding to 3S salt-extractable molecules. In castrated preparations the mean saturation levels (pmol/mg DNA) were different in the three organs: seminal vesicle, 2.8; ventral prostate, 1.8; caput epididymis, 0.9. The saturation level was significantly lower in ventral prostate of hypophysectomized rats (1.2) treated with testosterone to regenerate the accessory sex organs. Testosterone was the major nuclear androgen in the testes of mature hypophysectomized preparations perfused with testosterone. Although there was a large amount of nonspecific accumulation, testosterone binding to 3S molecules was shown by sucrose gradient centrifugation. Binding of dihydrotestosterone to 3S molecules in testicular nuclei was also demonstrated. The ratio of dihydrotestosterone to testosterone was different in immature and mature testicular nuclei and was altered by treatments known to affect testicular 5 alpha-reductase activity. The results suggest that in rat accessory sex organs and immature testis the major active androgen is dihydrotestosterone, whereas in mature testis it is testosterone. The shift in the predominant nuclear androgen in the testis from dihydrotestosterone to testosterone is most simply explained by the maturational change in 5 alpha-reductase activity.