Hippocampal neurons are activated during endurance exercise; however, little attention has been given to the location and spatial distribution of these neurons. We have, therefore, used Fos protein expression to identify the location and distribution of hippocampal neurons that become activated during acute moderate aerobic exercise. Adult rats were assigned into trained running (TR), trained nonrunning (TNR), untrained nonrunning (UNR), and cage-bound (CB) groups. Rats in the TR and TNR groups were trained to run, for three 20-min running periods separated by 3 min rest, on a treadmill. Rats in the UNR group spent identical time on a nonactivated treadmill, while rats in the CB group remained in their home cages throughout the training and experimentation. After training to criterion performance for both TR and TNR groups, both groups were rested for 1 day. Rats in the TR were then run on the treadmill to criterion level, while those in TNR and UNR groups spent equivalent time on the nonactivated treadmill. Animals in all groups were then killed and their brains removed, sectioned, and processed for Fos protein immunocytochemistry. Fos-like immunoreactive (FLI) neurons were counted in the dentate and CA1-3 fields of the hippocampus. The total numbers of hippocampal FLI neurons, as well as FLI neurons in each hippocampal region, were compared among groups. The total numbers of FLI neurons in the hippocampus, as well as in individual regions, were significantly greater in the TR group compared with the other three groups. Similarly, significant differences were found between the TNR group when compared with UNR and CB groups. Conversely, a significant difference existed between UNR and CB only in the CA1 field, which may account for the significant difference in the total number of hippocampal FLI neurons between these two groups. These results show that Fos induction occurs in the hippocampus during moderate physical exercise. Furthermore, the importance of the incorporation of adequate controls to account for possible differences in expression of immediate early gene expression due to trained performing, trained nonperforming, and untrained groups is discussed. The results indicate that adequate control for nonexercise stimuli is necessary for studies of the effect of exercise on the brain when expression of immediate early genes such as c-fos is used as an outcome measure.