ABC transporters like P-glycoprotein are the main membrane proteins responsible for drug transport in human cells and tumor tissue. This study was undertaken to investigate the effect of metabolic pathways on the P-glycoprotein expression in prostate adenocarcinoma cells DU145, Glioma cells Gli36 and KB-tumor cells which where transfected with a MDR-EGFP fusion gene. All cell lines were cultivated as multicellular tumor spheroids (MTS). Prostate MTS and Glia MTS developed with increasing diameter and time of culture a shift from aerobic to anaerobic glycolysis indicated by increasing lactate levels and decreasing pyruvate levels. With increasing lactate and decreasing pyruvate content in the cells the P glycoprotein synthesis was downregulated.Inhibiting glycolysis in general by iodoacetate (IA) and 2-desoxy-D-glucose (2-DDG) leads to a stimulated ROS generation and a downregulation of P-glycoprotein synthesis. The coincubation of MTS with IA or 2-DDG and the radical scavenger ebselene suppressed the ROS generation and the P-glycoprotein down-regulation. We propose that the radical scavenger properties of pyruvate - which is generated in large but non-necrotic MTS - conserves the P glycoprotein expression. Switching to lactate synthesis, the level of pyruvate is decreased and the ROS generation is stimulated leading to a downregulation of P glycoprotein expression. Co-incubation of MTS with IA, 2-DDG and the radical scavenger ebselene did not change the P-glycoprotein expression compared to control MTS. This observation points out that ROS are responsible for the decrease in P-glycoprotein expression. Additionally, IA and 2-DDG functionally inhibited the P-glycoprotein drug efflux by ATP depletion. This effect could not be reversed by radical scavengers.