Rat adipose tissues contain high-affinity, hormone-specific cytoplasmic estrogen receptors. If adipose tissues are actually estrogen target tissues, then it should be possible to demonstrate in vivo binding and retention of 17 beta-[2H]estradiol in adipose tissue cell nuclei, the putative subcellular site of steroid action. Gonadectomized rats were given an intravenous injection of 40 microCi (0.11 microgram) 17 beta-[2,4,6,7-3H]estradiol. Animals were then killed, and cell nuclei were isolated from hypothalamus and various fat pads. Cell nuclear levels of radioactivity peaked at 1 h in both hypothalamus and parametrial adipose tissue, but the peak cell nuclear concentration in the fat pad was more than twice that in hypothalamus. At 1 h, 89% of the adipose tissue nuclear radioactivity migrated with 17 beta-estradiol on thin-layer plates. Radioactivity was retained in cell nuclei for more than 6 h in both tissues. This cell nuclear binding was estrogen specific. Pretreatment with radioinert estrogens (17 beta-estradiol or R 2858) abolished cell nuclear 17 beta-[3H]estradiol uptake, but other steroids (progesterone, corticosterone, and 5 alpha-dihydrotestosterone) were without effect. Cell nuclear binding was found in all fat pads studied in both males and females. Levels were highest in parametrial (females) and epididymal (males) fat pads, the depots with the highest concentration of cytoplasmic estrogen receptor. There were no sex differences in cell nuclear binding. Taken together these findings provide strong support for the contention that 17 beta-estradiol could affect lipid metabolism and body fat content in part by direct action on adipose tissues.