In contrast to nonprimate species, the RNA for human tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, can undergo alternative splicing to produce four different types of mRNA. Although types 1 and 2 of these human tyrosine hydroxylase mRNAs have been identified in human brain, whether types 3 and 4 human tyrosine hydroxylase mRNAs are present in the central nervous system remains controversial. Furthermore, little is known about the expression of the protein products of these mRNAs in human brain. In this study we used antibodies raised against different octapeptide sequences from each of the predicted human tyrosine hydroxylase protein forms to determine the presence and distribution of each human tyrosine hydroxylase isoforms in several regions of human brain. Control immunocytochemical and blot immunolabeling experiments demonstrated that each antibody selectively recognized the human tyrosine hydroxylase isoform against which it was directed. In immunocytochemical studies, all four human tyrosine hydroxylase isoforms were clearly detectable in neurons of both the substantia nigra and locus coeruleus. The presence of all four isoforms in these nuclei was confirmed with blot immunolabeling studies. Single-label immunocytochemical studies of adjacent sections as well as dual-label comparisons of immunoreactivity for human tyrosine hydroxylase type 1 with type 2, type 3, or type 4 suggested that at least some neurons in these brain regions contain all four human tyrosine hydroxylase isoforms. In contrast, some neurons of the mesencephalon appeared to be selectively immunoreactive with the antibodies against type 1. In the caudate nucleus and putamen, the terminal zones of the dopaminergic projection from the substantia nigra, all four isoforms were detected, although in immunocytochemical studies type 1 appeared to be the predominant isoform present in axons and terminals. These findings demonstrate that human brain contains four distinct isoforms of human tyrosine hydroxylase and that the presence or relative amount of each isoform may differ among catecholaminergic cell populations and between catecholaminergic neurons and terminal fields. These patterns of expression may have important implications for understanding the regulation of catecholamine biosynthesis in human brain both in normal and pathological states.