Proteasome inhibitor bortezomib interacts with the regulation of protein turnover in eukaryotic cells. The results of proteasomal inhibition consist in cell cycle arrest and induction of apoptosis. Etoposid targets topoisomerase IIα and is included in several protocols for myeloma treatment. In this study, we examined the effects of simultaneous and sequential treatments of bortezomib and the topoisomerase IIα inhibitor etoposide in multiple myeloma cell lines, particularly in terms of potential synergistic effects between both drugs. Using the MTT assay, cytotoxicity levels for dosages ranging from 0.01 nM and 100 nM for bortezomib and 0.001 μM and 100 μM for etoposide in multiple myeloma cell lines OPM-2, NCI-H929 and RPMI-S were determined and IC50 values calculated. In each experiment, cells were treated with each drug individually and with fixed ratios of both drugs simultaneously (co-incubation over 48 h) and sequentially (pre-incubation with etoposide for 24 h and co-incubation with bortezomib for additional 24 h). The data were analyzed using the median effect method of Chou and Talalay, whereas the combination indices (CI) were calculated for each level of cytotoxicity. A CI< 1 indicated synergy, a CI= 1 indicated additivity and a CI> 1 indicated antagonism. In the sequential treatment schedule, we found synergistic effects in all three cell lines, even at low single-agent cytotoxicity levels (fractional inhibition <50%). Synergistic action were found in RPMI-S with a CI-range between 0.08 and 0.9, in OPM-2 between 0.05 and 0.07 and in NCI-H929 between 0.65 and 0.86 in different doses of both drugs. Interestingly, when cells were treated simultaneously with both drugs, no synergistic effects were observed. On cellular level, we found cell cycle arrest in the G2-M phase of the cell cycle, when cells were treated with both drugs sequentially. Furthermore, we noticed a correlation between the etoposide-sensitivity and G2-M/S-fraction of the sample, indicating that prolonged drug action in the G2-M/S-phase might contribute to enhanced growth inhibitory effects. On sub-cellular level, the synergy was accompanied by diminished activation of transcriptional factor NF-κB, whereas bortezomib abrogated the etoposide-induced NF-κB up-regulation. This effect was accompanied by down-regulation of Bcl-2, an anti-apoptotic protein and transcriptional product of NF-κB. Furthermore, when cells were treated simultaneously with both drugs, we noticed a translocation of topoisomerase IIα from nucleus into the cytoplasm, where the enzyme remained inactive. In sequential treatment schedule, no translocation of topoisomerase IIα was observed, indicating that the drug target remained in its functional compartment. In conclusion, our data show strong synergistic effects between bortezomib and etoposide in appropriate treatment schedules at low drug concentrations in vitro, indicating that the drug combination might be useful for clinical trials in multiple myeloma patients.