First line therapy for non-small cell lung carcinoma (NSCLC) commonly includes combination therapy with a tubulin-binding agent (TBA) and a DNA-damaging agent. TBAs suppress microtubule dynamics by binding to the beta-tubulin subunit of alpha/beta-tubulin, inducing mitotic arrest and apoptosis. Up-regulation of class III beta-tubulin (betaIII-tubulin) has been implicated in clinical resistance in NSCLC, ovarian and breast tumors treated in combination with a TBA and DNA-damaging agent. To investigate the functional significance of betaIII-tubulin in resistance to both these classes of agents, small interfering RNA (siRNA) was used to silence the expression of this isotype in two NSCLC cell lines, NCI-H460 and Calu-6. Reverse transcription-PCR and immunoblotting showed that betaIII-siRNA potently inhibited the expression of betaIII-tubulin, without affecting the expression of other major beta-tubulin isotypes. Clonogenic assays showed that betaIII-siRNA cells were significantly more sensitive to TBAs, paclitaxel, vincristine, and vinorelbine, and for the first time, DNA-damaging agents, cisplatin, doxorubicin, and etoposide compared with controls. Cell cycle analysis of H460 betaIII-siRNA cells showed reduced accumulation at the G(2)-M boundary and an increase in the sub-G(1) population in response to TBA treatment compared with control cells. Importantly, betaIII-siRNA cells displayed a significant dose-dependent increase in Annexin V staining when treated with either paclitaxel or cisplatin, compared with controls. These findings have revealed a novel role for betaIII-tubulin in mediating response to both TBA and DNA-damaging agent therapy and may have important implications for improving the targeting and treatment of drug-refractory NSCLC.