The distribution of the Florida Burrowing Owl (Athene cunicularia floridana) has expanded from primarily rural areas in south-central Florida to include urban/suburban areas to the north, northwest, south, and southeast. While Florida Burrowing Owls still reside in rural areas, the majority of previous research and current management strategies focus on urban/ suburban populations. Determining differences in the owl's ecology between rural and urban/suburban areas can aid in creating effective statewide management strategies for this species. We compared the available prey and diet of Burrowing Owls in a rural and urban environment. Although, on the basis of analysis of pellets, insects and arachnids were the two most frequent food items at both the rural and urban sites, the diets at the two sites differed, largely because of greater consumption of avian prey in the urban habitat. This study is the first comparison of dietary and prey-availability differences between rural and urban Burrowing Owl populations.