A genomic clone for 25-hydroxyvitamin D(3)-1alpha-hydroxylase (1alpha-hydroxylase) was isolated from a mouse embryonic stem cell P1 genomic library. It contains nine exons spanning 4.8 kb from the transcriptional start site. All the intron insertion sites are identical to that of the human 1alpha-hydroxylase and human vitamin D(3) 25-hydroxylase genes. A polyadenylation signal AUUAAA that differs from the consensus sequence at the second residue was identified 16 bp downstream of the 3' end of the previously reported mouse cDNA. This element is the only common natural variant described. The 3' end of the gene was determined using a RACE technique. Three poly(A) addition sites were observed 12, 15 and 22 bases from the AUUAAA element. Such distances are in agreement with what is required for maturation of mammalian pre-mRNAs. This molecular cloning makes possible the generation of transgenic mice in order to further investigate the role and importance of the 25-hydroxyvitamin D(3)-1alpha-hydroxylase (CYP1alpha).