Despite overall successful application to multiple myeloma patients, clinical efficacy of the proteasome inhibitor bortezomib is typically challenged by primary and secondary resistance of unknown origin. So far, the potential impact of intracellular concentrations on drug efficacy of bortezomib and the influence of drug transporters are unknown. We determined cellular bortezomib kinetics in nine myeloma cell lines using ultrahigh-performance liquid chromatography coupled to tandem mass spectrometry. The potential influence of drug transporters on the uptake kinetics observed in these cell lines was investigated by testing substrate characteristics of bortezomib for several transporters in over-expressing model cells. Additionally, transporter mRNA expression was quantified in myeloma cell lines by real-time polymerase chain reaction (RT-PCR). All myeloma cells revealed an extensive intracellular bortezomib accumulation (47.5-183 ng/ml) exceeding extracellular concentrations (0.04-0.17 ng/ml) by more than factor 1,000. Only organic anion-transporting polypeptide 1B1 facilitated the uptake in over-expressing cells, however, to a negligible extent (factor 1.36). Bortezomib efflux via P-glycoprotein was confirmed by demonstrating reduced sensitivity (IC50 11.6 vs. 2.8 ng/ml) and intracellular concentrations (-56.1%) in over-expressing cells compared to controls. RT-PCR revealed a varying but overall weak transporter expression in the studied myeloma cells without any correlation to intracellular concentrations. Although principally valid as demonstrated in the P-glycoprotein over-expressing cell model, there was no significant correlation between intracellular concentrations and bortezomib efficacy in myeloma cell lines. Differences in intracellular concentrations in myeloma cell lines neither result from variable transporter expression nor represent the main factor determining bortezomib efficacy in vitro.