MTS are a valuable in vitro model to study gene expression and necrosis development under physiological hypoxia. This study was undertaken to investigate the effects of hypoxia and evolving necrosis on the expression profile of genes involved in glycolysis and detoxification by real-time PCR. We further determined the generation of lactate and pyruvate. Prostate MTS (Du 145) developed increasing gradients of pericellular oxygen tension with increasing diameter/time of culture. First appearance of central necrosis was detectable in a diameter range of 200 to 300 mm. With increasing age and size of MTS, a shift from aerobic to anaerobic glycolysis occured. This was determined through an increase in lactate and decrease in pyruvate release of the cells as well as an up-regulation of the gene expression of hexokinase (1.5-fold), pyruvate dehydrogenase (2-fold) and lactate dehydrogenase (3-fold). Detoxification enzymes (SOD, GST) were up- regulated with evolving hypoxia but down-regulated with the onset of central necrosis in MTS. Inhibiting glycolysis by iodoacetate/desoxyglucose resulted in earlier development of central necrosis and stimulated ROS generation. We propose that the radical scavenger properties of pyruvate, which is produced in aerobic metabolism, influences the gene regulation of detoxification enzymes and suppresses the generation of necrosis.