Normal male rats received six subcutaneous injections of 8.0 pmoles of tritiated 25-hydroxy vitamin D 3 ([ 3H]25(OH)D 3) or one intrajugular injection of 8.0 pmoles of high specific radioactivity [ 3H]-25(OH)D 3. Lipid extracts of several tissues including the reproductive organs were subjected to sephadex LH-20 chromatography to determine the tissue distribution of the injected material and of the in vivo produced dihydroxylated cholecalciferol metabolites. The nature of the putative 25(OH)D 3 and the 24,25-dihydroxy vitamin D 3 (24,25(OH) 2D 3) from epididymis tissue was confirmed by high performance liquid chromatography (HPLC). The epididymis levels of 24,25(OH) 2D 3 were considerably higher in the cauda epididymis compared to kidney and caput epididymis levels. The other metabolites levels in this tissue were similar to those determined in the kidneys. The amounts of the three metabolites found in all other tissues were well below the cauda epididymis or kidney levels. The findings suggest a possible physiological role for 24,25(OH) 2D 3 in the epididymis, and are also consistent with data of others which indicated a possible action of 1,25-dihydroxy vitamin D 3 (1,25(OH) 2D 3) in rat reprductive tissues.