To elucidate molecular mechanisms involved in physical activity-induced beneficial effects on brain function, we studied in rats the influence of voluntary running on the activation in the hippocampus of cyclic AMP response element-binding protein (CREB) and mitogen-activated protein kinase (MAPK)/extracellular signal-regulated protein kinase (ERK). These are signaling molecules that play critical roles in synaptic plasticity, including learning and memory. Exercise resulted in an increase in the level of the activated transcription factor, CREB phosphorylated at Ser-133. The amount of the activated transcription factor about doubled already after 1 night of running and remained elevated for at least a week, although control levels were restored after 1 month of exercise. In addition, binding activity in nuclear extracts to cyclic AMP response element (CRE) motif containing oligonucleotides increased significantly in the hippocampus after 3 nights of exercise, although the total amount of the immunochemically identified CREB remained unaltered. Electrophoretic mobility supershift assays indicated that the increased binding was due to the recruitment of members of this transcription factor family, in addition to the CREB proper. Voluntary running also resulted in an increase in the level of phosphorylated MAPK (both p42 and p44). The time-courses of the increases in the level of the phosphorylated protein kinase and the activated transcription factor were different. In comparison with the activated CREB, the increase in the phosphorylated MAPK was delayed, but lasted longer, being detectable even after 1 month of exercise. These observations are consistent with the view that the relatively long-lasting activation of these signaling molecules participates in the regulation of genes, such as the neurotrophin genes, and contributes to the beneficial effects of physical exercise on brain function.