Deterioration of the hippocampus occurs in elderly individuals with and without dementia, yet individual variation exists in the degree and rate of hippocampal decay. Determining the factors that influence individual variation in the magnitude and rate of hippocampal decay may help promote lifestyle changes that prevent such deterioration from taking place. Aerobic fitness and exercise are effective at preventing cortical decay and cognitive impairment in older adults and epidemiological studies suggest that physical activity can reduce the risk for developing dementia. However, the relationship between aerobic fitness and hippocampal volume in elderly humans is unknown. In this study, we investigated whether individuals with higher levels of aerobic fitness displayed greater volume of the hippocampus and better spatial memory performance than individuals with lower fitness levels. Furthermore, in exploratory analyses, we assessed whether hippocampal volume mediated the relationship between fitness and spatial memory. Using a region-of-interest analysis on magnetic resonance images in 165 nondemented older adults, we found a triple association such that higher fitness levels were associated with larger left and right hippocampi after controlling for age, sex, and years of education, and larger hippocampi and higher fitness levels were correlated with better spatial memory performance. Furthermore, we demonstrated that hippocampal volume partially mediated the relationship between higher fitness levels and enhanced spatial memory. Our results clearly indicate that higher levels of aerobic fitness are associated with increased hippocampal volume in older humans, which translates to better memory function. Copyright 2008 Wiley-Liss, Inc.