APEX nuclease (Apex gene product) is a mammalian multifunctional DNA repair enzyme possibly involved in the repair of apurinic/apyrimidinic (AP) sites and single-strand DNA breaks with 3' termini blocked by nucleotide fragments and also in transcriptional regulation via redox activation of the AP-1 transcription factors. We cloned a 15-kb DNA fragment containing the Apex gene from a mouse leukocyte genomic library and determined a 4-kb stretch of its nucleotide sequence, including the complete sequence of the mouse Apex gene. The gene consists of 5 exons and 4 introns spanning 2.21 kb, and the boundaries between exons and introns follow the GT/AG rule. Two major and one minor transcription initiation sites were assigned to positions +1 and +24 and position +14, respectively, by a combination of ribonuclease protection, primer extension, and 5' RACE analyses. Position +1 is located 312 nucleotides upstream from the ATG initiation codon. The translation initiation and termination sites are located in exon II and exon V, respectively. The sequenced 5' flanking region (1.32 kb) lacks a typical TATA box, but contains a CAAT box and putative binding sites for several transcription factors, such as ATF, NF-IL6, Sp1, and AP2. The 0.8-kb region from position -410 (5' flanking region) to position +386 (intron II) contains a CpG island. The Apex gene locus was mapped to mouse chromosome 14C2-D1 using in situ hybridization.