We have identified and cloned a cDNA encoding a new member of the monooxygenase family of enzymes. This novel enzyme, which we call MOX (monooxygenase X; unknown substrate) is a clear sequence homologue of the enzyme dopamine beta-hydroxylase (DBH). MOX maintains many of the structural features of DBH, as evidenced by the retention of most of the disulfide linkages and all of the peptidyl ligands to the active site copper atoms. Unlike DBH, MOX lacks a signal peptide sequence and therefore is unlikely to be a secreted molecule. The steady-state mRNA levels of MOX are highest in the kidney, lung, and adrenal gland, indicating that the tissue distribution of MOX is broader than that of DBH. Antisera raised to a fusion protein of MOX identifies a single band of the expected mobility by Western blot analysis. MOX mRNA levels are elevated in some fibroblast cell strains at replicative senescence, through this regulation is not apparent in all primary cell strains. The gene for MOX resides on the q arm of chromosome 6 and the corresponding mouse homolog has been identified.