Multidrug resistance (MDR) mediated by the ATP-binding cassette proteins P-glycoprotein (Pgp), multidrug resistance protein (MRP-1), breast cancer resistance protein (BCRP) and the vault protein lung resistance protein (LRP) is implicated in treatment failure in acute myeloid leukemia (AML). Pgp, MRP-1 and BCRP mediate energy-dependent cellular drug efflux, while LRP blocks cytoplasmic-nuclear drug transport. MDR modulators are non-cytotoxic drugs that block the activity of MDR proteins. In clinical trials, the Pgp modulator cyclosporine A (CsA) improved treatment outcome in AML, while its non-immunosuppressive, non-nephrotoxic analogue PSC-833 has not, despite being a potent Pgp modulator in preclinical models. CsA is known to also modulate MRP-1, and we hypothesized that a broad spectrum of MDR modulation might contribute to its clinical efficacy. We studied the effects of CsA and PSC-833 on in vitro drug uptake, retention and cytotoxicity in drug-selected and transfected resistant cell lines overexpressing Pgp, MRP-1 or BCRP, and on nuclear-cytoplasmic drug distribution and cytotoxicity in cell lines overexpressing LRP. Cellular drug content was measured by flow cytometry, and nuclear-cytoplasmic drug distribution was assessed by confocal microscopy. CsA enhanced uptake and retention of the substrate drug mitoxantrone in cells overexpressing Pgp (HL60/VCR), MRP-1 (HL60/ADR) and BCRP (8226/MR20) and increased cytotoxicity 7-, 4- and 4-fold, respectively. Moreover, CsA increased the nuclear content of doxorubicin in 8226/MR20 cells, which co-express LRP with BCRP, and increased doxorubicin cytotoxicity 12-fold. The effect of CsA on nuclear drug content and cytotoxicity in 8226/MR20 cells occurred without an effect on cellular doxorubicin content, consistent with the fact that 8226/MR20 cells co-express wild type BCRP (BCRPR482), which does not efflux doxorubicin. Moreover the BCRP modulator fumitremorgin C had no effect on 8226/MR20 content, nuclear uptake nor cytotoxicity of doxorubicin. CsA also enhanced nuclear doxorubicin content in a second cell line with LRP-mediated resistance, HT1080/DR4. PSC-833 enhanced mitoxantrone retention and cytotoxicity in cells overexpressing Pgp, but, in contrast to CsA, had no effect on mitoxantrone uptake, retention nor cytotoxicity in cells expressing MRP-1 nor BCRP, and had no effect on doxorubicin nuclear content nor cytotoxicity in cells expressing LRP. Thus CsA is a broad-spectrum MDR modulator, with activity against Pgp, MRP-1, BCRP and LRP, while PSC-833 only modulates Pgp. The broad-spectrum activity of CsA may contribute to its clinical efficacy. These findings support identification and testing of other broad-spectrum MDR modulators.