The microsomal cytochrome P450 catalyzing the first step in the metabolic activation of vitamin D3 into its hormonal form 1 alpha,25-dihydroxyvitamin D3 has earlier been purified from pig liver. The present communication describes the cloning, structure, and expression of a cDNA encoding pig liver microsomal vitamin D3 25-hydroxylase. DNA sequence analysis of the cDNA revealed a 25-hydroxylase protein of 500 amino acids with a predicted molecular weight of 56,374. The structure of vitamin D3 25-hydroxylase, as deduced by both DNA sequence analysis of the cDNA and protein sequence analysis, shows 70-80% identity with members of the CYP2D family. Transfection of the vitamin D3 25-hydroxylase cDNA into simian COS cells resulted in the synthesis of an enzyme that was recognized by a monoclonal antibody raised against purified vitamin D3 25-hydroxylase and catalyzed 25-hydroxylation in the bioactivation of vitamin D3. Northern blot analysis showed that the mRNA for vitamin D3 25-hydroxylase is found in liver and kidney.