An antagonistic interaction between retinol and calciferol has been established. However, the mechanism by which this antagonism occurs is unclear. One possibility is that retinol affects the metabolism of calciferol. To investigate this hypothesis, retinol- and calciferol-depleted rats were given various amounts of ergocalciferol, cholecalciferol, 1alpha,25-dihydroxycholecalciferol [1,25(OH)2D3], or 24,24-difluoro-1alpha,25-dihydroxycholecalciferol [24-F2-1,25(OH)2D3] in combination with various amounts of retinyl acetate or all-trans retinoic acid (ATRA) in a series of studies. Rats administered 1720 or 3440 microg retinyl acetate once every 3 d for 33 d in combination with 25.8 ng ergocalciferol or 25 ng cholecalciferol every 3 d had lower serum calcium and greater serum phosphorus concentrations than rats fed 0 or 11.4 mug retinyl acetate every 3 d. In addition, rats fed 400 microg ATRA/d in combination with 25.8 ng ergocalciferol every 3 d, 25 ng cholecalciferol every 3 d, 2-5 ng 1,25(OH)2D3/d, or 0.5-1 ng 24-F2-1,25(OH)2D3/d had significantly lower serum calcium and higher serum phosphorus concentrations than rats not given ATRA in the diet. Therefore, both retinyl acetate and ATRA are able to antagonize the action of ergocalciferol and cholecalciferol in vivo. Additionally, ATRA antagonizes the in vivo action of 1,25(OH)2D3 and an analog, 24-F2-1,25(OH)2D3, that cannot be 24-hydroxylated. Together, these results suggest that retinol does not antagonize the action of calciferol by altering the metabolism of calciferol or 1,25(OH)2D3, but does so by another mechanism.