The relationship between potency of phenothiazine and naphthalene-sulfonamide calmodulin inhibitors and their effects on cellular levels and cytotoxic activity of doxorubicin was evaluated using the doxorubicin-sensitive and greater than 100-fold doxorubicin-resistant P388 mouse leukemia model system. In cytotoxicity studies using cell counts based on proliferation following a 24-hr drug exposure and in survival based on colony formation in soft-agar after a 2-hr drug exposure, the calmodulin inhibitors significantly enhanced the cytotoxic effects of doxorubicin in the resistant but not parent-sensitive P388 cells. However, survival in soft-agar (based on colony formation) following long-term drug exposure (approximately 120 hr) revealed that the cytotoxic effects of doxorubicin were significantly increased by the calmodulin inhibitors in both sensitive and resistant P388 cells. Laser flow cytometry studies on single-cell doxorubicin levels indicated that treatment with doxorubicin in the presence of trifluoperazine had no effect on drug levels in sensitive cells but significantly enhanced cellular accumulation and retention of doxorubicin in resistant cells. Furthermore, unlike treatment with doxorubicin alone, in the presence of trifluoperazine, heterogeneity in cellular drug levels in the resistant P388 cells was not observed. Among the various calmodulin inhibitors effective in enhancing cellular levels and cytotoxic effects of doxorubicin in the resistant P388 cells, chlorpromazine was approximately two-fold less potent than trifluoperazine or prochlorperazine and only N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide but not N-(4-aminobutyl)-2-naphthalenesulfonamide was active.