Genomic and overlapping cDNA clones encompassing the entire 5′-untranslated region of the human D5 receptor gene were cloned and sequenced. Comparison of these human D5 receptor genomic and cDNA clones revealed the presence of two exons separated by a small and variably sized intron (of either 179 or 155 bp). We have determined that the major site of transcription initiation of the D5 gene is 2125 bp upstream from the translational initiation start site. The region 5′ to the transcription initiation site lacked conventional TATA and CAAT sequences, but contained several putative binding sites for transcription factors, such as Spl and Apl. Luciferase reporter gene constructs containing D5 gene sequence information up to 500 bp 5′ of the transcription initiation site were able to stimulate transcription only in SK-N-SH cells but not in COS-7, CHO, P19EC, NB41A3, and SK-N-MC cell lines. Promoter deletion analysis indicated that the D5 gene promoter contained a positive modulator at 119-182 and a negative modulator 251-500 bases upstream from the site of transcription initiation. In addition, in order to detect the expression of functional D5 receptor mRNAs and not those of its expressed pseudogenes, in situ hybridization analysis of monkey and human brain using a 5′ D5-specific riboprobe revealed that D5 receptor mRNA was most abundant in discrete cortical areas (layers II, IV, and VI), the dentate gyrus, and hippocampal subfields with very little message detected in the striatum. Unexpectedly, D5 mRNA antisense riboprobes labeled discrete cell bodies in the pars compacta of the substantia nigra. The characterization of the genomic organization of the D5 receptor gene and of those factors involved in its transcriptional regulation may aid in our understanding of the role this gene product plays in the generation and maintenance of dopamine D1-like receptor-mediated events. © 1995, American Chemical Society. All rights reserved.