Intramuscular administration of 1,25 (R or S),26-trihydroxyvitamin D3 to dairy cows resulted in significant elevation of 1,25-dihydroxyvitamin D3 concentration in blood plasma. The increase of 1,25-dihydroxyvitamin D3 in plasma paralleled the increase of 1,25,26-trihydroxyvitamin D3. We also found that intramuscular 1,25-dihydroxyvitamin D3 administration to dairy cows significantly elevated plasma 1,24,25-trihydroxyvitamin D3. These data suggest possible impairment of 1,25-dihydroxyvitamin D3 metabolism during the presence of pharmacologic concentrations of 1,25,26-dihydroxyvitamin D3 in plasma. In addition, the 24-hydroxylation of 1,25-dihydroxyvitamin D3 to 1,24,25-trihydroxyvitamin D3 appears to be an important route for further metabolism of 1,25-dihydroxyvitamin D3 in dairy cows.