The mechanism of chemotherapy resistance in breast cancer is unresolved. MDR1/P-glycoprotein (P-Gp) over-expression confers multidrug resistance in vitro and might play a role in clinical breast cancer. Studies using clinical samples have yielded conflicting results. MDR1/P-Gp mRNA expression was determined relative to the expression in normal human liver using TaqMan real-time RT-PCR (corrected for expression of the housekeeping gene PBGD). Immunohistochemistry (IHC) was performed with monoclonal antibodies against P-Gp (JSB1, C219). The positive control was SW1573/ 2R160, the intermediate control SW1573 and the negative control GLC4/ADR. We assayed 9 breast-cancer cell lines by RT-PCR and IHC, 52 carcinoma samples by RT-PCR and 168 samples by IHC. SW1573/2R160 contained high levels of MDR1/P-Gp mRNA (1.0, equal to liver) and showed strong membranous staining. Expression of MDR1/P-Gp mRNA in SW1573 (0.05) and GLC4/ADR (3.2 Ψ 10 -5 ) was not detectable by IHC. Very low levels of MDR1/P-Gp mRNA were measured in breast-cancer cell lines (mean 3.1 Ψ 10 -4 , range 1 to 12 Ψ 10 -4 ), but P-Gp was not detected by IHC. In 25 specimens from chemotherapy-naive patients, MDR1/P-Gp mRNA levels varied from 1 to 11 Ψ 10 -2 (mean 3.9 Ψ 10 -2 ). In sections of 80 chemotherapy-naive tumors, no membranebound staining was observed in the tumor cells. Tumors of 27 anthracycline-treated patients had comparable MDR1/P-Gp mRNA expression levels (mean 5.4 Ψ 10 -2 ). P-Gp was undetectable in 88 tumor samples of patients who had received anthracycline-based chemotherapy. In breast cancer, MDR1/ P-Gp mRNA is low or absent and P-Gp levels in cancer cells are too low to detect by IHC. Chemotherapy exposure does not result in detectable MDR1/P-Gp over-expression.