Epithelial-specific Ets (ESE) transcription factors, consisting of ESE-1, ESE-2, and ESE-3, are constitutively expressed in distinct epithelia of mucosal tissues, including the lung. Each ESE member exhibits alternative splicing and yields at least two isoforms (a and b) with transcriptional targets largely unidentified. The studies described herein define a novel role for ESE transcription factors in transactivation of the human lysozyme gene (LYZ), an essential component of innate defense in lung epithelia. Of the six ESE isoforms, ESE-1a and ESE-1b transactivated LYZ promoter in reporter gene assays, while only ESE-1b dramatically upregulated transcription of endogenous LYZ in both nonpulmonary and pulmonary epithelial cells. Importantly, ESE-1a and ESE-1b could transactivate the LYZ promoter in cultured primary airway epithelial cells. ESE-2 and ESE-3 isoforms were unable to substantially transactivate the lysozyme promoter or up-regulate transcription of endogenous LYZ. Two functional consensus Ets sites located in the proximal 130bp LYZ promoter were responsive to ESE-1b as identified by site-directed mutagenesis and DNA binding assays. Short hairpin RNA (shRNA) attenuation of endogenous ESE-1b mRNA levels in lung epithelia resulted in decreased LYZ transcription. Furthermore, ESE-1 antibody specifically enriched the 130bp proximal LYZ promoter in chromatin immunoprecipitation analyses. These findings define a novel role for ESE transcription factors in regulating lung innate defense and suggest distinct regulatory functions for ESE family members.