Transcription of the rat alpha 1B adrenergic receptor (alpha 1BAR) gene in the liver is controlled by three promoters that generate three mRNAs. The middle promoter (P2), located between -432 and -813 base pairs upstream from the translation start codon and lacking a TATA box, is responsible for generating the major, 2.7-kilobase mRNA-species expressed in many tissues (Gao, B., and Kunos, G. (1994) J. Biol. Chem. 269, 15762-15767). DNase I footprinting using rat liver nuclear extracts identified three protected regions in P2: footprint I (-432 to -452), footprint II (-490 to -540), and footprint III (-609 to -690). Putative response elements in footprints I and III were not analyzed except the AP2 binding site in footprint III, which could be protected by purified AP2 protein. Footprint II contains four sites corresponding to half of the NF-I consensus sequence, but DNA mobility shift assays indicate that this footprint binds two proteins distinct from NF-I: a ubiquitous CP1-related factor and another novel factor, termed alpha-Adrenergic Receptor Transcription Factor (alpha ARTF), which binds to two separate sites in this region. The alpha ARTF is widely distributed, with the highest amounts found in brain, followed by liver, kidney, lung, and spleen, but no detectable activity in heart. Deletions of alpha ARTF binding sites nearly abolished P2 promoter activity, which suggests that the alpha ARTF is essential for the transcription of the alpha 1BAR gene in most tissues.