Diet composition of the Barn Owl Tyto alba was studied in agricultural landscapes in Thessaly, Greece, for 3 years (2003–05). A total of 852 Rattus spp. individuals were identified from 10 065 pellets, which accounted for 2.9% by frequency and 27.4% by biomass of 29 061 prey items. Rattus spp. were more numerous in Barn Owl pellets during winter months than in summer. We suggest that this difference was due to a shift in relative prey availability and an increased need for energy by the Barn Owl during the colder months. Comparisons between Thessaly and 15 other areas showed differences in prey availability between islands and mainland Greece. Microtus spp. were absent from all island diets except one, and some islands had greater species richness.