The steroid hormone 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] produces biological responses by interaction with both a well-characterized nuclear receptor (VDR(nuc)) to regulate gene transcription and with an as-yet uncharacterized membrane-associated protein/receptor (VDR(mem)) to generate a variety of rapid, non-genotropic responses. We report for the first time that [3H]1alpha,25(OH)(2)D(3) binds with high affinity to a chick duodenal caveolae-enriched membrane fraction (CMF) isolated without the use of detergents. Caveolae are plasma membrane invaginations implicated in signal transduction and molecular transport processes. Using the CMF fraction as a possible source of VDR(mem), we found that the in vitro binding of [3H]1alpha,25(OH)(2)D(3) was ligand dependent and saturable; the K(D) and B(max) were 1.3+/-0.6nM and 29+/-11fmol 1,25(OH)(2)D(3)/mg protein (n=17), respectively. Immunoblot analysis of the CMF confirms the presence of caveolin-1, a marker protein for membranes with caveolae. Therefore, chick CMF may represent a good source for isolation and characterization of the putative VDR(mem) for 1alpha,25(OH)(2)D(3).