Overexpression of anti-apoptotic Bcl-2 family members and deregulation of the pathways that regulate pro-apoptotic family members have been observed in non-small cell lung cancers (NSCLC). Previous reports have identified both Bcl-2 and Bcl-x(L) proteins as survival factors in lung cancer cells since reductions in these proteins can induce apoptosis and sensitize lung cancer cells to apoptosis induced by chemotherapy agents. Myeloid cell leukemia-1 (Mcl-1), another member of the Bcl-2 family, has been found to be a critical survival factor in hematopoietic cells, yet little data exists for a role of Mcl-1 in human lung cancers. We used NSCLC cell lines to explore how Mcl-1 levels affect lung cancer cell survival and studied tumors from patients to determine expression patterns of Mcl-1. NSCLC cells express abundant Mcl-1 protein and depletion of Mcl-1 levels by antisense Mcl-1 oligonucleotides induces apoptosis in A549 and H1299 lung cancer cells. Reduction in Mcl-1 levels can sensitize lung cancer cells to apoptosis induced by cytotoxic agents as well as by ionizing radiation. Lung cancer cells overexpressing Mcl-1 are less sensitive to apoptosis induced by chemotherapeutic agents, ZD1839 (an inhibitor of EGFR tyrosine kinase) and Bcl-2 or Bcl-x(L) antisense oligonucleotides. We find that epidermal growth factor (EGF) can enhance Mcl-1 protein levels in an ERK-dependent manner. Signal transduction agents that reduce Mcl-1 levels correlated with their individual ability to induce apoptosis in lung cancer cells. Finally, NSCLC tumors taken directly from patients have elevated levels of Mcl-1 protein compared with normal adjacent lung tissue. Therefore, agents that target Mcl-1 can induce apoptosis and sensitize cells to apoptosis induced by cytotoxic agents. Mcl-1 protein is overexpressed in a subset of human NSCLC and enhanced levels of Mcl-1 may protect lung cancer cells from death induced by a variety of pro-apoptotic stimuli.