Calmodulin inhibitors enhance cytotoxic effects of doxorubicin (DOX) in DOX-resistant (P388/DOX) P388 mouse leukemia cells by increasing cellular accumulation and retention of drug. In P388/DOX cells treated for 3 hr, cytotoxic effects (based on colony formation in soft agar) of daunorubicin (DAU) in the presence of trifluoperazine (TFP) were DAU concentration-dependent and enhanced 2- to 100-fold. Additionally, in the presence of TFP, on a molar basis, equitoxic doses of DAU were 4-fold lower than DOX for P388/DOX cells. However, in P388/DOX cells treated for 3 hr with other anthracyclines, except for a slight enhancement in the cytotoxic effects of aclacinomycin A (ACM) with TFP, colony formation in soft agar of cells treated with N-trifluoroacetyladriamycin-14-valerate (AD32) and N-trifluoroacetyladriamycin were similar in the absence and presence of TFP. In DOX-sensitive (P388/S) P388 mouse leukemia cells treated for 3 hr, some enhancement in the cytotoxic effects due to TFP were observed with DAU and DOX but not with ACM, AD32, or N-trifluoroacetyladriamycin. Although accumulation of ACM and AD32 in P388/S and P388/DOX cells was similar and unaffected by TFP, the retention of ACM but not AD32 was enhanced 1.5-fold only in TFP-treated P388/DOX cells. In contrast, DAU accumulation in P388/S cells was 4-fold higher than in similarly treated P388/DOX cells, and the 2- and 4-fold increase due to TFP in the accumulation and retention, respectively, of DAU in P388/DOX cells was not observed in P388/S cells. Results from this study indicate that in P388/DOX cells, the calmodulin inhibitor TFP is more effective with DAU than DOX, significantly less effective with ACM, and ineffective with AD32 and N-trifluoroacetyladriamycin.