Interactions between vitamin A and vitamin D have been suggested for several decades but have not been established. In particular, vitamin A has been proposed to intensify the severity of the bone mineralization disease, rickets and inhibit the ability of vitamin D to cure this disease. To investigate this hypothesis, weanling Holtzman rats were fed a 1.2% calcium, 0.1% phosphorus diet and 15.5 ng ergocalciferol (vitamin D(2)) every 3 d for 21 d in the presence of increasing amounts of retinyl acetate (0 microg to 8621 microg/d). The increasing amounts of retinyl acetate produced a progressive and significant decrease in total bone ash (P < 0.001) and an increase in epiphyseal plate width (P < 0.001). The same experiment conducted with increasing amounts of vitamin D(2) (0 to 645 ng/d) indicated that the antagonism by retinyl acetate could be demonstrated at all vitamin D(2) dosages. To further investigate this antagonistic relationship, weanling Holtzman rats were fed a 0. 47% calcium, 0.3% phosphorus diet and 15.5 ng vitamin D(2) every 3 d for 33 d in the presence of increasing retinyl acetate (0 to 3448 microg/d). In the absence of retinyl acetate, these rats maintained a normal serum calcium level (2.34 mmol/L). Increasing retinyl acetate, however, eliminated the ability of vitamin D(2) to elevate the level of serum calcium (1.35 mmol/L). These results illustrated in vivo antagonism of vitamin D(2) action on intestine and bone by retinyl acetate.