Licensed London taxi drivers show that humans have a remarkable capacity to acquire and use knowledge of a large complex city to navigate within it. Gray matter volume differences in the hippocampus relative to controls have been reported to accompany this expertise. While these gray matter differences could result from using and updating spatial representations, they might instead be influenced by factors such as self-motion, driving experience, and stress. We examined the contribution of these factors by comparing London taxi drivers with London bus drivers, who were matched for driving experience and levels of stress, but differed in that they follow a constrained set of routes. We found that compared with bus drivers, taxi drivers had greater gray matter volume in mid-posterior hippocampi and less volume in anterior hippocampi. Furthermore, years of navigation experience correlated with hippocampal gray matter volume only in taxi drivers, with right posterior gray matter volume increasing and anterior volume decreasing with more navigation experience. This suggests that spatial knowledge, and not stress, driving, or self-motion, is associated with the pattern of hippocampal gray matter volume in taxi drivers. We then tested for functional differences between the groups and found that the ability to acquire new visuo-spatial information was worse in taxi drivers than in bus drivers. We speculate that a complex spatial representation, which facilitates expert navigation and is associated with greater posterior hippocampal gray matter volume, might come at a cost to new spatial memories and gray matter volume in the anterior hippocampus. © 2006 Wiley-Liss, Inc.