Exogenous ATP activates purinoreceptors on the cell surface that regulate diverse cellular functions, including mucous cell secretion in the respiratory epithelium. In this study, ATP increased MUC5AC mRNA in primary human nasal epithelial cells and in NCI-H292 pulmonary adenocarcinoma cells in vitro. ATP-induced MUC5AC mRNA was mediated by phospholipase Cbeta3. A dominant-negative mutation in the PDZ binding domain of PLCbeta3 inhibited ATP-induced MUC5AC gene expression. ATP sequentially activated the phosphorylation of Akt, ERK1/2, p38, RSK1, and cAMP-responding element-binding protein (CREB) in a protein kinase C-independent manner. ATP-induced MUC5AC mRNA levels were regulated by CREB via direct interaction with c-Ets1 on the MUC5AC gene promoter (located -938 to -930). Effects of CREB and c-Ets1 were additive. Inhibition of either CREB or c-Ets1 inhibited ATP-induced MUC5AC gene expression. Stimulation with ATP caused the direct binding of CREB and c-Ets1 to the MUC5AC promoter, increasing the phosphorylation of c-Ets1. Chromatin immunoprecipitation assays demonstrated that in the presence of ATP, both c-Ets1 and CREB bound to the MUC5AC promoter. The effects of exogenous ATP on MUC5AC gene expression are mediated by a complex regulatory cascade controlling interactions between CREB and c-Ets1 that bind to a promoter element in the MUC5AC gene enhancing MUC5AC gene transcription. ATP-dependent activation of MUC5AC gene expression via CREB-c-Ets1 may contribute to mucous cell hypersecretion associated with common respiratory disorders.