The distribution of specific binding sites for vitamin D3 in adult female and male Xiphophorus helleri is studies after injection of tritiated 1,25-dihydroxyvitamin D3 (vitamin D) by thaw-mount autoradiography. Five hours after injection of labeled vitamin D specific nuclear binding is present in brain, pituitary, skin, gills, cartilage, gut, liver, pancreas, spleen, kidney, muscle, ovary, and testis. Cytoplasmic binding exists strongest in gills, gut, and kidney while it is comparatively weak in hepatocytes. In reproductive organs cytoplasmic retention of radioactivity is also present in oocytes. Weak nuclear labeling exists in interstitial cells in ovary. Conspicuous nuclear labeling exists in active lobules of testis, while inactive lobules show occasionally a few labeled cells. The results demonstrate specific binding and retention of vitamin D in many target organs of teleost fish, suggesting an extensive and multifunctional regulatory role of this steroid hormone of sunlight.