Tyrosine hydroxylase catalyzes the rate-limiting reaction in the biosynthesis of the catecholamine neurotransmitters and hormones (dopamine, norepinephrine, and epinephrine). Rat tyrosine hydroxylase exists, in its native form, as a tetramer composed of identical 498 amino acid subunits. There is currently no information describing the molecular interactions by which the four monomeric tyrosine hydroxylase subunits assemble into an active tetramer. Mutational analysis was performed on bacterially expressed enzyme to assess the role of a putative C-terminal leucine zipper in the assembly of subunits into the tetrameric holoenzyme. Deletion of the C-terminal 19 amino acids, or mutation of a leucine residue (to an alanine), converts the enzyme from a tetrameric to a dimeric form that exhibits greater structural heterogeneity. This change in macromolecular form is accompanied by a 75% (deletion mutation) to 20% (Leu-->Ala mutation) reduction in specific activity of the enzyme. This represents the first report of the functional involvement of a region containing a leucine zipper motif in the assembly and activity of a neuronal enzyme.