The overexpression of P-glycoprotein (PGP) and alterations in DNA topoisomerase II (TOPO II) were evaluated in mouse leukemia P388 cells selected in vivo for mitoxantrone (MTT) resistance (P388/MTT) and compared to doxorubicin (DOX) resistant (P388/DOX) or vincristine (VCR) resistant (P388/VCR) models. Among a panel of TOPO II inhibitors which included etoposide (VP-16), DOX, MTT and 4'-[(9-acridinyl)-amino]methanesulfon-m-anisidide (m-AMSA), the relative resistance compared to parental sensitive P388/S cells was: P388/DOX greater than P388/MTT greater than P388/VCR. All the resistant sublines exhibited minimal cell kill (less than 20%) at vincristine concentrations greater than 100-fold the IC50 for P388/S cells. In a soft-agar colony-forming assay, the modulation of cytotoxicity in P388/MTT cells by the calmodulin inhibitor trifluoperazine following a 3-hr drug treatment demonstrated a marked potentiation in cell kill with MTT, VP-16, DOX and m-AMSA but not VCR. Immunoblotting data revealed that while PGP was not detectable in P388/S cells, the overexpression of PGP was apparent in P388/MTT cells and the relative expression between the resistant sublines was: P388/DOX greater than P388/MTT greater than P388/VCR. Although the amount and DNA cleavage activity of TOPO II in nuclear extracts from P388/VCR cells were comparable to those in P388/S cells, they were markedly lower in both P388/DOX and P388/MTT cells. However, decatenation activity of TOPO II in nuclear extracts was comparable between the sensitive (P388/S) and resistant sublines (P388/MTT, P388/DOX, and P388/VCR). Results from the present study demonstrated that P388 cells selected for resistance to mitoxantrone exhibit changes in TOPO II and overexpression of PGP similar to P388/DOX cells, while vincristine resistant cells only overexpress PGP. Since therapeutic strategies are primarily designed to interfere with PGP-mediated drug efflux, the choice of agents for modulating resistance in tumors which overexpress PGP versus tumors which overexpress PGP with altered TOPO II could be different.