Ets proteins play a vital role in the regulation of mammalian immunity, and family members Ets-1 and Ets-2 regulate a variety of genes that participate in the propagation of an inflammatory response. Heme oxygenase (HO)-1, although acutely induced by inflammatory stimuli, has cytoprotective properties and prevents an exaggerated inflammatory response. Ets-1 and Ets-2 both induce HO-1 promoter activity; however, Ets-2 was a more potent transactivator of HO-1 in macrophages. A potent inflammatory mediator, bacterial lipopolysaccharide (LPS), induced Ets-2 at the mRNA and protein level, and this induction preceded the up-regulation of HO-1. To further delineate the role of Ets-2 in regulating HO-1 transcription, we performed HO-1 promoter analysis studies in macrophages. Deletion mutants down to -137/+74 maintained an activity analogous to that of the largest construct, -4045/+74. Further deletion constructs (starting with -117/+74) showed a significant reduction in promoter activity when co-transfected with Ets-2 or exposed to LPS. Promoter sequence analysis revealed two putative Ets binding sites (EBSs) in this region, and mutation of these sites showed that EBS -93, more than EBS -125, was critical for full HO-1 promoter activity. Additional studies showed that EBS -93 binds Ets-2 and that mutation of the DNA binding domain of Ets-2 entirely prevented transactivation of HO-1. Finally, overexpression of a dominant negative form of Ets-2 blunted HO-1 promoter induction by LPS, and kinase inhibitors (PI3K more than JNK) that reduced Ets-2 expression markedly decreased endogenous HO-1 expression. Our data provide evidence that Ets-2 contributes to the up-regulation of HO-1 by the potent inflammatory stimulus LPS in macrophages.