The aryl hydrocarbon receptor (AhR) is a mediator of xenobiotic toxicity, best recognized for conveying the deleterious effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure. The AhR functions as a ligand-activated transcription factor that binds to a canonical xenobiotic response element (XRE) in association with the heterodimerization partner, the AhR nuclear translocator (Arnt) protein. However, within the repertoire of AhR target genes identified in recent years, many lack a clearly defined XRE highlighting the growing realization that AhR-mediated gene expression seems to involve additional mechanisms distinct from the well characterized process involving the XRE. The present study characterized a novel nonconsensus XRE (NC-XRE) in the promoter of the plasminogen activator inhibitor-1 (PAI-1) gene that recruits a novel protein-DNA complex responsible for TCDD-inducible expression. DNA binding studies and reporter assays identified key residues in the NC-XRE necessary for protein-DNA binding and function, respectively. Functional studies with AhR expression constructs confirm that TCDD-inducibility is AhR-dependent and requires direct AhR-DNA binding to the NC-XRE. Chromatin immunoprecipitation and RNA interference studies reveal that the Arnt protein is not a component of the NC-XRE-bound AhR complex, suggesting that in contrast to the XRE, AhR-dependent gene expression mediated through the NC-XRE may involve a new DNA binding partner.