mRNA for dopamine beta-hydroxylase [3,4-dihydroxyphenylethylamine, ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1] has been partially purified from poly(A)+ mRNA isolated from a rat pheochromocytoma cell line. Shared antigenic determinants between tyrosine hydroxylase and dopamine beta-hydroxylase allowed us to obtain enriched fractions of dopamine beta-hydroxylase mRNA by immunoprecipitating translated mRNA products with tyrosine hydroxylase antisera. The enriched dopamine beta-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 beta-hydroxylase. Partial proteolysis of both authentic dopamine beta-hydroxylase and the protein encoded by the recombinant clone produced identical peptide patterns.