We have previously shown marked induction of the stress-inducible gene heme oxygenase-1 (HO-1) in vivo and in vitro after hyperoxia. In RAW 264.7 cells, HO-1 induction is transcriptionally regulated and dependent on cooperation between the HO-1 gene promoter and the 5' distal enhancer element SX2. In our present study, further deletional and mutational analyses demonstrate that signal transducer and activator of transcription (STAT) DNA binding sites located in the promoter of HO-1 and activator protein (AP)-1 DNA binding sites in the distal enhancer element SX2 are necessary for optimal HO-1 gene activation after hyperoxia. Interestingly, a second 5' distal enhancer element, AB1, located 10 kb upstream from the HO-1 promoter, alone is activated after hyperoxia but cannot confer maximal hyperoxia-induced HO-1 gene transcription. Mutational analysis of the AB1 enhancer shows that AP-1 is essential for AB1-mediated HO-1 gene transcription after hyperoxia. Electromobility shift assays show increased STAT1, STAT3, STAT5, and AP-1 DNA binding activity in RAW 264.7 cells after hyperoxia. Taken together, our data suggest that the 5' distal enhancer elements of the HO-1 gene in concert with the promoter regulate HO-1 gene induction and highlight the complexity of HO-1 gene transcription in response to hyperoxia.