The physiological significance of 24R,25-dihydroxyvitamin D3 [24R,25 (OH)2D3] is still controversial. The effects of 24R,25-(OH)2D3 on serum calcium level were examined in rats. While 24R,25-(OH)2D3 had no effect on serum calcium level in normal rats, 200 micrograms/kg of 24R,25-(OH)2D3 showed significant hypocalcemic effect in rats pretreated with vitamin D3 (changes of serum calcium level compared with control group were 1.3 and 1.5 mg/dl at 24 and 36 hours respectively) and also showed a significant antagonistic effect against the action of 1 alpha,25-dihydroxyvitamin D3 (1 alpha,25(OH)2D3) in the thyroparathyroidectomized rats (changes of serum calcium compared with control group were 0.5 and 0.8 mg/dl at 12 and 24 hours respectively). It was concluded that the effect of 24R,25-(OH)2D3 on serum calcium differs from that of 1 alpha,25-(OH)2D3.