From 2004 to 2006 we studied the spatial activity of Long-eared Owls (Asio otus) breeding in the city of České Budějovice, Czech Republic. Two main factors (size and proportion of home range in developed area) clearly distinguished the owls into two groups: urban (n = 5) and suburban (n = 4). Urban owls occupied home ranges >50% in developed area; home ranges of suburban owls were <50% in developed area. We obtained 866 radio fixes from nine individuals while they were feeding offspring, when both males and females participated in hunting. The home ranges of urban owls were larger than those of suburban owls (95% kernels: 446 vs. 56 ha, respectively). Urban males and females used home ranges of similar sizes that overlapped greatly with those of the opposite sex and with those of other breeding pairs. In contrast, suburban male owls had home ranges larger than those of suburban females. Wooded areas and meadows were the vegetation types preferred by both urban and suburban owls, and both groups avoided vegetation types associated with human activities. Six of nine owls preferred the edges of open habitats, such as meadows and fields bordering wooded areas. Urban owls used remnant green spaces (refugia) within the city area to hunt small mammals. Long-eared Owls use nests made by other species, and may profit in urban environments from increased nest sites due to the relatively recent colonization of urban areas by the Common Magpie (Picapica) and from suitable hunting grounds within the city.