mRNA for dopamine β-hydroxylase [3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (β-hydroxylating), EC 126.96.36.199] has been partially purified from poly(A)+ mRNA isolated from a rat pheochromocytoma cell line. Shared antigenic determinants between tyrosine hydroxylase and dopamine β-hydroxylase allowed us to obtain enriched fractions of dopamine β-hydroxylase mRNA by immunoprecipitating translated mRNA products with tyrosine hydroxylase antisera. The enriched dopamine β-hydroxylase mRNA was used to synthesize the corresponding cDNAs, which were then cloned in the Pst I site of pBR322. Recombinant colonies were characterized by an in situ colony immunoassay and hybrid-selected translation. In vitro translation of the mRNA selected from one recombinant clone produced a protein of 75,000 daltons that comigrated with authentic dopamine β-hydroxylase. Partial proteolysis of both authentic dopamine β-hydroxylase and the protein encoded by the recombinant clone produced identical peptide patterns.