Thymocytes induce their glycolytie enzymes as they undergo transition from the resting to the proliferating state. Corresponding increases in mRNA levels point to a transcriptional regulation. Electrophoretic mobility shift assays revealed that the DNA-binding efficiency of Spl is increased when nuclear extracts from proliferating compared to resting rat thymocytes were used. Here we demonstrate that hydrogen peroxide, added to nuclear extract from proliferating cells, decreases the Spl DNA-binding activity, whereas in nuclear extracts from resting cells dithioerythritol fully restores DNA-binding efficiency. Moreover we show that in contrast to resting thymocytes, production of reactive peroxide anions upon priming with phorbol 12-myristate 13-acetate is nearly abolished in the proliferating cells. From these results we propose that reactive oxygen intermediates affect the interaction of the Spl transcription factor with its consensus sequence and subsequently regulate glycolytic gene expression.