Parvalbumin (PV) is a calcium-binding protein of the EF-hand family, expressed mainly in fast contracting/relaxing muscles of vertebrates. We have isolated five overlapping genomic PV clones which overall span 28 kilobase pairs (kb) around the Pva locus on mouse Chromosome (Chr) 15. The positions of four introns were determined by DNA sequencing. They interrupt the coding sequences at positions corresponding to those in rat and human PV genes. The transcription start site, 25 bp downstream from the TATA-box, was mapped by oligonucleotide primer extension on poly(A)(+)-RNA. The analysis of 0.4 kb promoter sequence of the mouse PV gene revealed CCAAT- and TATA-box sequences and a 59 bp GC-rich stretch between positions -59 and -118. Similar motifs have been found in the parvalbumin genes of rat and human. A perfect 11-bp repeat upstream to positions -149 and -163 respectively is homologous only to the rat promoter. These results will be related to tissue and species differences in PV expression.