Cytosolic receptor-like binders for 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃] have been demonstrated in several mammalian tissues, including intestine, kidney, and bone. To establish which other organs in the body possess the receptor and thus to elucidate new potential target tissues of 1,25- (OH)₂D₃, we thought it of interest to examine numerous mammalian organs for specific 1,25-(OH)₂D₃ receptor-like binders. Cytosols from 13 murine tissues were prepared using hypertonic buffer and were incubated for 3 h at 0 C with 1,25-(OH)₂[³H]D₃. When examined on 5–20% sucrose density gradients, cytosols from kidney, intestine, bone, skin, and mammary gland exhibited a 3.2S peak which was specific for 1,25-(OH)₂D₃ and presumably represents the 1,25-(OH)₂D₃ receptor. Brain, lung, thymus, adipose tissue, skeletal muscle, diaphragm, uterus, and myocardium lacked a detectable 3.2S peak, but displayed varying amounts of an approximate 6S peak, presumably representing the binding of 1,25-(OH)₂[³H]D₃ to the ubiquitous 25-hydroxyvitamin D₃ plasma-generated binder. Biochemical measurements of specific binding were employed using charcoal or hydroxylapatite techniques to separate bound from free steroids. Competitive binding studies showed those tissues with only the 6S binder to have preferential binding of 25-hydroxyvitamin D₃ and those tissues possessing a 3.2S peak to have preferential binding of 1,25- (OH)₂D₃. Scatchard analyses of kidney, bone, skin, and intestinal cytosols revealed a single class of noninteracting binding sites with an equilibrium dissociation constant (Kd) of approximately 0.2 nM for each tissue. Our findings correlate with published data on the tissue distribution of vitamin D-dependent calcium-binding protein. In summary, we have demonstrated the absence of a specific 1,25-(OH)₂D₃ 3.2S receptor-like macromolecule in a variety of mammalian organs. Furthermore, in addition to the previously known organs ie, intestine, bone, and kidney, we have elucidated two new potential 1,25-(OH)₂D₃ target organs, skin and mammary gland. The binder has an identical sedimentation constant and Kd in each tissue, suggesting that the receptor is the same in every tissue in which it is present.