We have isolated and characterized genomic DNA clones encoding rabbit phospholamban. Only a single gene for phospholamban was detected in the rabbit genome. The phospholamban gene of 13.2 kilobases contains only one 10.5-kilobase intron, which separates exonic sequences located in the 5'-untranslated region. Two potential transcription initiation sites were mapped to 335 and 185 nucleotides upstream from the translation initiation site in the mRNA or 239 and 89 nucleotides upstream from the exon/intron boundary. Phospholamban gene expression in various smooth muscle tissues, as well as in cardiac and slow twitch skeletal muscle, was detected by Northern blotting. The phospholamban gene was mapped to human chromosome 6, using a human phospholamban cDNA.