Three overlapping cholesterol 7 alpha-hydroxylasecDNAs were cloned from total mouse liver RNA by a combination of the polymerase chain reaction and mouse liver cDNA library screening. One of the cDNA clones was used to screen a mouse genomic library; three genomic clones were isolated and one of them was fully characterized. The mouse cholesterol 7 alpha-hydroxylase gene spans approximately 10 kb. The gene contains six exons including a 1509-bp open reading frame encoding 503 amino acid residues. The predicted amino acid sequence of mouse cholesterol 7 alpha-hydroxylase showed 93 and 82% identity to the rat and human enzymes, respectively. The transcription initiation site is mapped to 64 bases 5' of the translation initiation codon. Several transcription factor binding sequences are identified in the 5' flanking region of the mouse cholesterol 7 alpha-hydroxylase gene.