Purpose: P-glycoprotein is a 170-kDa plasma membrane multidrug transporter that actively exports cytotoxic substances from cells. Overexpression of P-glycoprotein by tumor cells is associated with a multidrug-resistant phenotype. Immunosuppressive agents such as cyclosporins and macrolides, have been shown to attenuate P-glycoprotein activity. However, the mechanism by which some immunosuppressants inhibit P- glycoprotein function has not been determined. Since cyclosporin and macrolide immunosuppressants inhibit calcineurin (CaN) phosphatase and FKBP12 peptide-prolyl isomerase (FKBP12 PPI) activity, studies were conducted to determine if these effects are directly related to the inhibitory effects these immunosuppressants have on P-glycoprotein function. Methods: Western blot analysis was performed to assess CaN and FKBP12 protein levels in P- glycoprotein-negative (MCF-7) and -positive (MCF-7/Adr) breast cancer cell lines. P-glycoprotein function was determined by intracellular doxorubicin accumulation and/or cytotoxicity assays before and after CaN and FKBP12 were independently inhibited by pharmacological antagonists. Results: CaN and FKBP12 levels were similar in MCF-7 and MCF-7/Adr cells. P-glycoprotein function was not affected by treatment of P-glycoprotein-expressing MCF- 7/Adr cells with CaN and FKBP12 antagonists. Conclusions: These results demonstrate that the inhibitory effects of immunosuppressive agents on P- glycoprotein function are independent of CaN or FKBP12 PPI activity.