Six mature, nonlactating, nonpregnant Jersey cows were treated with five 1-alpha-hydroxylated metabolites of vitamin D3 to compare relative potencies of the metabolites. Blood plasma 1,25-dihydroxyvitamin D peaked at about 1000 pg/ml 12 h after intramuscular injection of 500 micrograms 1,25-dihydroxyvitamin D3 but returned to preinjection concentration within 3 to 4 days. Administration of 500 micrograms 1-alpha-hydroxyvitamin D3 intramuscularly led to a gradual increase in 1,25-dihydroxyvitamin D concentration to 500 pg/ml 48 h after treatment. When 500 micrograms of 1,25-dihydroxyvitamin D3 or 1 alpha-hydroxyvitamin D3 were given orally, plasma 1,25-dihydroxyvitamin D peaked at about 200 and 100 pg/ml, respectively. The decline from peak concentrations occurred more slowly after oral treatment than after intramuscular treatment. Plasma calcium was increased by 20 to 30% for a week or more, depending on 1,25-dihydroxyvitamin D concentrations in plasma. All treatments resulted in calciuria. Treatment with 1 or 3 mg doses of 1,24(R),25-trihydroxyvitamin D3 or both carbon-25 stereoisomers of 1,25,26-trihydroxyvitamin D3 induced smaller and less protracted changes in plasma minerals.