Resistance to the cytotoxic effects of daunomycin and Adriamycin (ADR) in sublines of Ehrlich ascites and P388 mouse tumors has been demonstrated to be due to reduced cellular accumulation and retention of drug. In this study, the effect of the calmodulin inhibitor trifluoperazine on the cellular accumulation, retention, and cytotoxic effects of ADR in ADR-sensitive (P388/S) and ADR-resistant (P388/R) P388 mouse leukemia cells was determined. In cells treated in suspension culture for 24 hr or for 1 hr followed by plating in soft agar, a noncytotoxic concentration of 4 microM trifluoperazine, enhanced the sensitivity to ADR 2- to 6-fold in P388/R but not in P388/S cells. A marked enhancement in cellular retention rather than accumulation of ADR in only P388/R cells was obtained with trifluoperazine treatment. This study suggests the possible novel use of phenothiazines to improve drug sensitivity of tumors resistant to ADR treatment.