Large-conductance, voltage-dependent and Ca2+-sensitive K+ (BK) channels are composed of pore-forming α subunits and the modulatory β subunits. In smooth muscle, the modulatory β1 subunits are vital in rendering BK channels function as an important regulator of smooth muscle tone and excitability. In this study, we cloned and characterized the BK β1 subunit gene from rabbits (New Zealand white) and observed its tissue distribution pattern. The full-length cDNA of the BK β1 subunit, amplified by 5′-RACE and 3′-RACE, is 1,437 bp in nucleotide containing a 447 bp 5′-UTR, a 385 bp 3′-UTR and a 576 bp open reading frame (ORF) which encodes a peptide of 191 amino acids. Sequence analyses showed that the rabbit BK β1 subunit cDNA is 90, 84 and 82% homologous with that of human, mouse and rat respectively. The similarity is 86, 83, and 83% at the deduced amino acids level with human, mouse and rat β1 subunit gene, respectively. Northern blots indicated that the rabbit BK β1 subunit gene is highly expressed in sphincter of Oddi (SO) and aortal smooth muscle tissues, whereas with relatively lower level of expression in heart and skeletal muscle tissues and with no expression found in tissues of liver, lung, kidney and brain. Bioinformatics analyses indicated that the encoded protein is a membrane protein with two transmembrane helical regions containing four functional domains, one possible PKA phosphorylation site (T14) at the N-terminal and two N-glycosylation sites (N80 and N142) at the extracellular loop. For the first time, we identified and characterized the full-length cDNA sequence of the rabbit BK channel β1 subunit gene, which will set the basis for further investigation in the transcriptional regulation of this gene.