A number of cDNA clones for rat dopamine beta-hydroxylase (DBH) were isolated from a rat pheochromocytoma tumor cDNA library. The 2445 nucleotide sequence revealed a single open reading frame of 1860 nucleotides and a 3' untranslated region containing two polyadenylation addition signals. The cDNA coded for a 620 amino acid protein of 69,883 daltons. Six potential glycosylation sites and one potential phosphorylation site were identified. Amino acid residues likely to be involved in the active site of DBH and in copper ligand binding were identified. The N-terminal 42 amino acids appeared to constitute a typical but unusually long signal sequence. Hydropathy analysis indicated that this N-terminal region contained the only extensive hydrophobic domain and thus constituted the only obvious potential membrane attachment site. Northern analysis detected two mRNA species of 2.5 and 2.7 kb. The relative abundance of the 2.7 vs. 2.5 kb mRNAs was differentially regulated in PC12 cells and adrenals. DBH mRNA levels were induced in vivo in rat adrenals upon treatment with reserpine.