The effect of pyridoxal 5'-phosphate on the 1, 25-dihydroxyvitamin D3 receptor system has been studied by using pig intestinal chromatin. Pyridoxal 5'-phosphate did not affect the binding of 1, 25-dihydroxyvitamin D3 to its receptor extracted from chromatin with hypertonic KCI, although in the presence of pyridoxal 5'-phosphate 1, 25-dihydroxyvitamin D3-receptor complexes were not readily precipitated with polyethylene glycol. In contrast, pyridoxal 5'-phosphate showed a potency to dissociate the 1, 25-dihydroxyvitamin D3 receptor from chromatin in a dose-dependent manner. A low concentration of pyridoxal 5'phosphate was as effective as hypertonic KCl in dissociating the receptor from chromatin, while pyridoxine, p-nitrophenyl phosphate, or inorganic phosphate was much less effective. These observations suggest the inhibitory effect of pyridoxal 5'-phosphate on the recognition of 1, 25-dihydroxyvitamin D3 by its receptor system.