A beta 1-adrenergic receptor (beta 1AR) clone, designated Clone 5, was isolated from a BALB/c mouse liver genomic library screened at low stringency with a human brain beta 2 AR cDNA probe. Sequence analysis of Clone 5 revealed a 1,395-bp open reading frame encoding a 464-amino-acid polypeptide. The predicted protein exhibited structural features characteristic of members of the G-protein-coupled receptor family including seven hydrophobic segments corresponding to putative transmembrane domains, a potential N-linked glycosylation site near the amino-terminus, and multiple potential phosphorylation sites in the third cytoplasmic loop and carboxy-terminal cytoplasmic tail. The sequence of the Clone 5-encoded protein was nearly identical to those previously reported for the rat and human beta 1 ARs. Potentially important differences were noted in the third cytoplasmic loop and carboxy-terminal cytoplasmic tail. Reverse transcription-primer extension studies of adult mouse brain RNA demonstrated the predominant transcriptional start site to be 415 nucleotides upstream of the translational start site. A GC-box precedes the transcriptional start site by 40 nucleotides. No consensus TATA or CAAT box sequences were identified in this region. Southern blot analysis of a Chinese hamster x mouse somatic cell hybrid panel and of the progeny of an inter-subspecies backcross mapped the Clone 5-encoded gene to mouse chromosome 19, the localization previously determined for the mouse homolog of the human beta 1AR gene. Binding studies of transient COS-7 transfectants and stable L-cell transfectants confirmed that Clone 5 encodes a beta AR of the beta 1 subtype. A probe derived from Clone 5 selectively hybridized in Northern blot studies to mRNA isolated from adult mouse cerebrum, lung, and heart. These data should serve as the basis for further studies of the regulation and function of the beta 1AR.