The overexpression of P-glycoprotein (P-gp) is associated with multidrug resistance (MDR) of tumor cells to a number of chemotherapeutic drugs. P-gp inhibitors have been shown to effectively reverse P-gp-mediated MDR in both in vitro and in vivo. Our previous studies demonstrated that E6, a novel synthetic calmodulin inhibitor, exhibited potent inhibitory effect on P-gp in rat brain microvessel endothelial cells (RBMECs). In the present study, the effect of E6 on MDR in a K562 MDR cell line (K562/DOX) highly expressing P-gp was studied and compared with that of a conventional P-gp inhibitor, verapamil (VER). E6 at concentrations of 1, 3, 10, 30 microM reduced the IC50 value of doxorubicin in K562/DOX cells from 79.19 microM to 35.18, 21.86, 6.31 and 1.97 microM, respectively. However, the IC50 value of doxorubicin in K562 sensitive subline was not significantly changed by E6. Using a DNA content analysis and an annexin V binding assay, the effects of E6 on doxorubicin-induced apoptosis were also examined. The results indicated that E6 effectively reversed the resistance to doxorubicin-induced apoptosis in K562/DOX cells. In addition, co-treatment of E6 and doxorubicin resulted in a remarkably G2/M blocking effect in K562/DOX cells. Furthermore, the treatment of K562/DOX cells with 10 microM E6 led to increased intracellular accumulation and decreased efflux of doxorubicin. Overall, the pharmacological effects of E6 on P-gp-mediated MDR is much stronger than that of positive control drug VER. These results suggested that E6 is a novel and potent MDR reversal agent and may be a potential adjunctive agent for tumor chemotherapy.