IKKalpha, a subunit of IkBalpha kinase (IKK) complex, has an important role in the activation of nuclear factor-kB (NF-kB), a key regulator of normal and tumor cell proliferation, apoptosis, and response to chemotherapy. However, little is known about the transcriptional regulation of the IKKalpha gene itself. The present study revealed that the transcriptional induction of the IKKalpha gene is positively regulated by binding ETS-1, the protein product of the ETS-1 proto-oncogene. Furthermore, ETS-1 mediated activation of IKKalpha is negatively regulated by p53 binding to ETS-1. By analyzing the genomic DNA sequence, we identified the putative IKKalpha promoter sequence in the 5'-flanking untranslated region of the IKKalpha gene. Transfection of EU-4, an acute lymphoblastic leukemia (ALL) cell line, with plasmids containing the IKKalpha 5'-untranslated region sequence upstream of the luciferase reporter showed that this region possessed major promoter activity. Induction or enforced overexpression of p53 represses IKKalpha mRNA and protein expression as well as IKKalpha promoter activity. Deletion and mutation analyses as well as chromatin immunoprecipitation and electrophoretic mobility shift assay indicated that ETS-1 binds to the core IKKalpha promoter and strongly induces its activity. Although p53 does not directly bind to the IKKalpha promoter, it physically interacts with ETS-1 and specifically inhibits ETS-1-induced IKKalpha promoter activity. These results suggest that the proximal 5'-flanking region of the IKKalpha gene contains a functional promoter reciprocally regulated by p53 and ETS-1. Furthermore, loss of p53-mediated control over ETS-1-dependent transactivation of IKKalpha may represent a novel pathway for the constitutive activation of NF-kB-mediated gene expression and therapy resistance in cancer.