Microsomal epoxide hydrolase (EPHX1) catalyzes hydration reactions that determine the cellular disposition of reactive epoxide derivatives. Whereas the previously defined EPHX1 exon 1 sequence (E1) is derived from a promoter proximal to exon 2 of the EPHX1 coding region, in this investigation, we identified an alternative EPHX1 exon 1 sequence, E1-b, originating from a gene promoter localized approximately 18.5 kb upstream of exon 2. Northern hybridizations demonstrated that the E1-b variant is widely expressed and that the E1-b promoter functions as the primary driver of EPHX1 expression in human tissues. In contrast, the E1 promoter directs expression only in the liver. To examine the basis for liver-specific usage of the E1 promoter, we identified several potential cis-regulatory elements that included GATA (-110/-105) and hepatocyte nuclear factor 3 (HNF3) (-96/-88) motifs. GATA-4 was the principal GATA family member interacting with its respective motif, whereas both HNF3alpha and HNF3beta were capable of interacting with the HNF3 element. GATA-4 and HNF3alpha/HNF3beta DNA binding complexes were enriched in hepatic cells. Site-directed mutagenesis and transactivation analyses of the E1 promoter revealed that GATA-4 is probably a principal factor that regulates liver-specific expression of the E1 variant, with HNF3alpha and HNF3beta acting to negatively regulate GATA-4 function in hepatic cells.