Cytochrome P-450 3A4 (CYP3A4), the predominant cytochrome P-450 expressed in adult human liver, is subject to transcriptional induction by a variety of structurally unrelated xenobiotics, including the antibiotic rifampicin. The molecular mechanisms underlying this phenomenon are poorly understood. We transfected a human liver-derived cell line (HepG2) with various CYP3A4-luciferase reporter gene constructs containing a nested set of 5'-deletions of the CYP3A4 5'-flanking region. Rifampicin-inducible transcription of the reporter gene was observed only with the longest construct, which encompassed bases -13000 to +53 of CYP3A4 (3-fold induction). The responsive region was functional regardless of its position or orientation relative to the proximal promoter of CYP3A4 and was capable of conferring rifampicin-inducible expression on a heterologous promoter. Further deletion mutants localized the induction to bases -7836 to -7607. In vitro DNase I footprint analysis of this region revealed four protected sites (FP1, FP2, FP3, and FP4). Two of these sites, FP3 (bases -7738 to -7715) and FP4 (bases -7698 to -7682), overlapped binding motifs for the orphan human pregnane X receptor (hPXR). Cotransfection of responsive constructs with a hPXR expression vector substantially increased the rifampicin-inducibility to approximately 50-fold. In addition, the rifampicin-responsive constructs were strongly activated by a range of CYP3A inducers. Finally, we demonstrate cooperativity between elements within the distal enhancer region and cis-acting elements in the proximal promoter of CYP3A4. Our results provide evidence for the existence of a potent enhancer module, 8 kb distal to the transcription start point, which mediates the transcriptional induction of CYP3A4 by activators of hPXR.