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The metabolism of 25-hydroxycholecalciferol (25-(OH)D₃) was investigated in rats fed a diet low in calcium and without vitamin D for 4 weeks after hypophysectomy. Compared with intact rats on the same diet these animals had a low serum phosphorus concentration, a less marked degree of hypocalcaemia and their parathyroid gland was not hypertrophied. Eighteen hours after i.p. injection of a single dose of tritiated 25-(OH)D₃, chromatography of serum extracts on Sephadex LH-20 showed that the percentage of radioactivity corresponding to 1,25-dihydroxycholecalciferol (1,25-(OH)₂D₃) was lower in hypophysectomized rats than in control rats. High-pressure liquid chromatography demonstrated that 81% of this material had the same elution profile as synthetic 1,25-(OH)₂D₃. The percentage of 1,25-(OH)₂D₃ in the serum of hypophysectomized rats could be increased to the level seen in the controls by chronic treatment with bovine growth hormone. This action of growth hormone was most probably independent of the parathyroid glands since the injection of parathyroid extract did not alter 25-(OH)D₃ metabolism in hypophysectomized animals. These results suggest that the decrease in the conversion of 25-(OH)D₃ to 1,25-(OH)₂D₃ after hypophysectomy may be related to the lack of growth hormone.