The 14-3-3 proteins have a wide range of ligands and are involved in a variety of biological pathways. Importantly, 14-3-3 proteins are known to be overexpressed in some human lung cancers, suggesting that they may play a role in tumorigenesis. Here we examined 14-3-3 expression in several lung cancer-derived cell lines and found that four of the seven 14-3-3 isoforms, beta, epsilon, theta and zeta, were highly expressed in both lung cancer cell lines and normal lung fibroblasts. Two isoforms, sigma and gamma, were present only at very low levels. Immunoprecipitation data showed 14-3-3zeta could bind to CDC25C in irradiated A549 cells, and suppression of 14-3-3zeta in A549 cells with antisense resulted in a decrease in CDC25C localization in cytoplasm and CDC2 phosphorylation on Tyr15. As a consequence, CDC2 activity remained elevated which resulted in release from radiation-induced G(2)/M-phase arrest. Moreover, 16% 14-3-3zeta antisense-transfected cells underwent apoptosis when exposed to 10 Gy ionizing radiation. These data indicate that 14-3-3zeta is involved in G(2) checkpoint activation and that inhibition of 14-3-3 may be a useful approach to sensitize human lung cancers to ionizing radiation.