Altered vitamin D metabolism has been implicated as a cause of anticonvulsant-induced osteomalacia. Previous studies have demonstrated accelerated biotransformation of vitamin D3 to 25-hydroxycholecalciferol (25-OHD3). However, it is not known whether the 25-OHD3 is metabolized to 1, 25-dihydroxycholecalciferol (1, 25-(OH) 2D3), the tissue-active metabolite of vitamin D3. This study using rats was undertaken to study the influence of phenobarbital on the biotransformation of 25-OHD3 to 1, 25-(OH) 2D3. The disappearance rate of 25-OHD3 was virtually the same in the pheno-barbital-treated group (re= 0.0615 pmole/min) and the control group (re-0.0549 pmole/min). Similarly, the appearance rate of 1, 25-(OH) 2D3 was virtually the same in the treated group (ra= 0.0133 pmole/min) and the control group (ra= 0.0134 pmole/min). These data suggest that phenobarbital does not affect the biotransformation of 25-OHD3 to 1, 25-(OH) 2D3. The implication of this observation is that altered vitamin D metabolism does not account for phenobarbital-induced osteomalacia.