13.329 kilobases of the single copy human tyrosine hydroxylase (hTH) gene were isolated from a genomic library. The 5' flanking 11 kilobases fused to the reporter green fluorescent protein (GFP) drove high level expression in TH+ cells of the substantia nigra of embryonic and adult transgenic mice as determined by double label fluorescence microscopy. To provide a basis for future analysis of polymorphisms and structure-function studies, the previously unreported distal 10.5 kilobases of the hTH promoter were sequenced with an average coverage of 20-fold, the remainder with 4-fold coverage. Sequence features identified included four perfect matches to the bicoid binding element (BBE, consensus: BBTAATCYV) all of which exhibited specific binding by electrophoretic mobility shift assay (EMSA). Comparison to published sequences of mouse and rat TH promoters revealed five areas of exceptional homology shared by these species in the upstream TH promoter region -2 kb to -9 kb relative to the transcription start site. Within these conserved regions (CRs I-V), potential recognition sites for NR4A2 (Nurr1), HNF-3beta, HOXA4, and HOXA5 were shared across human, mouse, and rat TH promoters.