Voltage- and Ca²⁺-sensitive K⁺(MaxiK) channels play key roles in controlling neuronal excitability and vascular tone. We cloned and analyzed human and rodent genes for the modulatory β subunit, KCNMB1. The human and mouse β-subunit genes are ∼11 and ∼9 kb in length, respectively, and have a four exon–three intron structure. Primer extension assay localized the transcription initiation site at 442 (human) or 440 (mouse) bp upstream of the translation initiation codon, agreeing with the transcript size in Northern blots. Both genes have a TATA-less putative promoter region, with a transcription initiator-like region, and motifs characteristic of regulated promoters, including muscle-specific enhancing factors-1 and -2. Consistent with a tissue-specific expression of KCNMB1, regulated at the transcriptional level, β-subunit transcripts are abundant in smooth muscle and heart, but scarce in lymphatic tissues, brain, and liver. Expressed rat and mouse β subunits increase the apparent Ca²⁺sensitivity of the human MaxiK channel α subunit.