The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay is a widely used method in assessment of cytotoxicity and cell viability, and also in anti-cancer drug studies with tumour cells. These cells often express efflux proteins, such as P-glycoprotein (MDR1) or multidrug resistance (MDR) protein 1 (MRP1). MDCKII cells that overexpress these proteins (MDCKII-MDR1 or MDCKII-MRP1) and normal cells (MDCKII-wt) were used to investigate the effects of efflux pump activity on the results of MTT assay. Efflux protein activity was confirmed with calcein-AM efflux assay, and MTT assay was compared to another cytotoxicity test, the LDH release assay. Inhibition of MRP and MDR1 efflux proteins in MDCKII cell lines was associated paradoxically with increased reduction of MTT, implying an apparent increase in cell viability. This effect was seen when MK 571 (MRP1 and MRP2 inhibitor) or verapamil (MRP1 and MDR1 inhibitor) were used to block efflux protein activity. The calcein-AM efflux assay also showed that the MTT reagent inhibits the function of MDR1 in the MDCKII-MDR1 cell line. This study shows that MDR1 and possibly MRP proteins interfere with the MTT assay. Due to wide substrate specificity of efflux proteins and popularity of the MTT assay this interference is not trivial. Presence of any efflux protein substrate may therefore lead to underestimated results in MTT assay, thereby causing potential bias and erroneous conclusions in cytotoxicity studies.