Abstract 1,25-Dihydroxyvitamin D 3(1,25-(OH) 2D 3) receptor was solubilized in cytosol fractions upon homogenization of hen intestinal mucosa with pyridoxal 5'-phosphate contained in a low ionic strength buffer. Pyridoxal 5'-phosphate did not inhibit the binding of 1,25-(OH) 2D 3 to its receptor. The receptor solubilized with pyridoxal 5'-phosphate was similar to the KCl-solubilized receptor in its binding affinity to the hormone and sedimentation coefficient. A majority (>90%) of the mucosal 1,25-(OH) 2D 3 receptors were obtained as associating with crude chromatin which was prepared with a low ionic strength buffer, and this fraction of the receptor was solubilized with pyridoxal 5'-phosphate. Ten millimolar pyridoxal 5'-phosphate was as effective as approx 0.2 M KCl in solubilizing the receptor from the crude chromatin. Pyridoxal 5'-phosphate also showed a potency to dissociate the 1,25-(OH) 2D 3-receptor complex previously bound to DNA-cellulose. Pyridoxal 5'-phosphate-related compounds such as pyridoxamine 5'-phosphate and pyridoxal did not show this potency. These results suggest that pyridoxal 5'-phosphate reduced the interaction of 1,25-(OH) 2D 3 receptor with its nuclear binding components without inhibiting the binding of the receptor to the hormone.