The accumulation of <sup>3</sup>H-testosterone and its metabolites in the brain of neonatal and prepubertal rats was measured following injections of the hormone in vivo, or after incubation of minced brain tissue with <sup>3</sup>H-testosterone in tissue culture medium. Radioactivity associated with purified nuclei and various cytoplasmic fractions of brain tissue was determined in 2–5-day, 10–12-day, and 25–32-day-old animals. Comparisons of radioactive steroids, accumulated 2 h after s.c. injections of <sup>3</sup>H-testosterone or <sup>3</sup>H-estradiol, showed that testosterone (T) is accumulated to a lesser extent than estradiol (E), but the age-related patterns of uptake are similar for both steroids. A continuous decrease in radioactivity was observed in nuclear and cytoplasmic fractions from the brains of 2-, 4-, and 12-day-old animals that were given injections of <sup>3</sup>H-testosterone in vivo. This age-related pattern of decreasing uptake of radioactivity was not observed when excised brain tissue was incubated with <sup>3</sup>H-testosterone in tissue culture medium. Thus, it appears that the age-related responsiveness of neonatal rats to T may be due more to the fact that decreasing amounts of the hormone reach the brain in older animals than that an inherent difference exists in tissue susceptibility during this period. Most of the radioactivity accumulated in the brain was associated with cytoplasmic fractions. Less than 1% of the homogenate radioactivity accumulated in purified nuclei. This restricted uptake and the kinetics of <sup>3</sup>H-testosterone nuclear accumulation suggest that a nuclear site of action of T and/or its metabolites exists in the neonatal rat brain.