Carp (Cyprinus carpio), a freshwater fish that lives in a low-calcium environment, and Atlantic cod (Gadus morhua), a seawater fish that lives in a high-calcium environment, were studied for the presence of a novel membrane binding protein ("receptor") for the vitamin D metabolite, 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Basal lateral membranes from enterocytes of either species were prepared and analyzed for specific saturable binding. Membranes from carp revealed a dissociation constant of 1.23 nM with a maximal binding capacity of 212 fmol/mg protein. In comparison, membranes from Atlantic cod enterocytes revealed very low and nonsignificant levels of specific binding. The [³H]1,25(OH)₂D₃ binding activity in carp was further characterized for protein dependence, detergent extractability, and competition for binding with the metabolites 25(OH)D₃ and 24R,25(OH)₂D₃. Finally, introduction of 1,25(OH)₂D₃ to isolated carp enterocytes enhanced protein kinase C activity within 5 min, whereas intracellular Ca²⁺ concentrations were unaffected by a range of 1,25(OH)₂D₃ concentrations, as judged by fura 2 fluorescence. Thus the binding moiety may be a putative plasma membrane receptor for vitamin D, because it is functionally coupled to at least one signal transduction pathway.