We have shown that thyroid hormones have a direct effect on the renal metabolism of 25-hydroxyvitamin D3 (25OHD3). Using a new high performance chromatographic technique based upon a cyano-bonded packing, we studied the metabolism of [26,27-3H]25OHD3 in the isolated perfused rat kidney in vitro. We measured the rate of synthesis of 24,25-dihydroxyvitamin D3 [24,25-(OH)2D3], 24-oxo-25-hydroxyvitamin D3 [24-oxo-25OHD3], and 24-oxo-23,25-dihydroxyvitamin D3 [24-oxo-23,25-(OH)2D3] by the kidneys of vitamin D-replete [D(+)] rats and the rate of synthesis of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] by the kidneys of vitamin D-deplete [D(-)] rats. Synthesis of 24,25-(OH)2D3 increased significantly at 2 and 6 h of perfusion in perfused kidneys treated with T3, T4, and TSH as compared to untreated controls. Synthesis of 24-oxo-25OHD3 increased at 2 and 6 h and that of 24-oxo-23,25-(OH)2D3 increased at 6 h in T3- and TSH-treated groups. Perfused kidneys from vitamin D(-) rats treated with T3, T4, and TSH synthesized significantly decreased amounts of 1,25-(OH)2D3 at 2, 4, and 6 h. TRH treatment of kidneys from either vitamin D(+) or D(-) rats caused no significant changes in synthesis of any vitamin D metabolites when compared with appropriate controls. These results suggest T3, T4, and perhaps TSH, but not TRH, have a direct effect on renal 25OHD3 metabolism.