Non-small cell lung cancer cells (NSCLC) are more resistant to anticancer treatment as compared with other types of cancer cells. Recently (Hemström et al., Exp Cell Res 2005;305:200–13) we showed that apoptosis of U1810 NSCLC cells induced by the staurosporine analog PKC 412 correlated with inhibition of Akt and ERK1/2, suggesting the involvement of these kinases in cell survival. Here we investigated the contribution of the PI3-kinase/Akt and MEK/ERK pathways to survival of NSCLC cells. The two signaling pathways were studied by using different combinations of the PI3-kinase inhibitors LY-294002 and wortmannin, the Akt activator Ro 31-8220, the MEK inhibitor PD 98059 and PKC 412. PI3-kinase inhibitors induced apoptosis-like death in U1810 cells. H157 cells in general were relatively resistant to PI3 kinase/Akt inhibitors yet these compounds sensitized cells to the DNA-damaging drug VP-16, while Ro 31-8220 could not. PD 98059 only had a sensitizing effect on H157 cells when combined with PI3-kinase inhibition and VP-16. Morphological data indicated that LY-294002 and PKC 412 induced cell death at anaphase and metaphase, respectively, suggesting death by mitotic catastrophe. Analyzes of cells blocked in G2/M-phase by nocodazol revealed that LY-294002 increased, while PKC 412 decreased histone H3 phosphorylation, suggesting that LY-294002 allowed, while PKC 412 inhibited cells to leave M-phase. Flow cytometric analysis of cell cycle distribution demonstrated that LY-294002 allowed cells to leave G2/M phase, while PKC 412 inhibited cytokinesis, resulting in formation of multinucleated cells. These results indicate that sensitization of NSCLC cells by PI3-kinase inhibition involves interplay between cell cycle regulation, mitotic catastrophe and apoptosis. © 2006 Wiley-Liss, Inc.