Approximately 7% of a 650 pmol dose of 25-hydroxy[26,27-14C]vitamin D3 and 25% of a 325 pmol dose of 1,25-dihydroxy[26,27-14C]vitamin D3 are metabolized to 14CO-2 by vitamin D deficient rats. Nephrectomy prevents the metabolism of 25-hydroxy[26,27-14C]vitamin D3 to 14CO2 but not that of 1,25-dihydroxy[26,27-14C]vitamin D3. Less than 5% of the 14C from 24,25-dihydroxy[26,27-14C]vitamin D3 is metabolized to 14CO2. Feeding diets high in CA and supplemented with vitamin D3 markedly diminishes the amount of 14CO2 formed from 25-hydroxy[26,27-14C]vitamin D3 but not that from 1,25-dihydroxy[26,27-14C]vitamin D3. These results indicate that only 1-hydroxylated vitamin D compounds and especially 1,25-dihydroxyvitamin D3 undergo side-chain oxidation and cleavage to yield an unknown metabolite and CO2.