Abstract.—Flight initiation distance (FID) is the distance to which a predator is permitted to approach before the prey initiates flight behavior. This can be influenced by factors including predator density and distance to cover. We measured flight initiation distances in two populations of Western fence lizards (Sceloporus occidentalis), one in a rural and one in an urban environment. Lizards at the rural site initiated flight at significantly longer distances than those at the urban site. These results support the prediction that lizard behavior can be influenced by differences in human exposure and predator recognition in their environments. Lizards develop tolerance to humans in urban areas in order to maximize fitness by reserving energy and utilizing resources for longer periods of time. The findings from this study demonstrate that researchers should be aware of differences in human exposure between populations of study animals and should choose study sites appropriately when designing FID studies.