The diet of the Barn Owl was investigated in 13 localities in the Pisa province, Central Italy. In each locality the percentage of forest and cultivated land, and the linear development of roads and rivers were recorded. To estimate the prey availability, the small mammal communities in seven different localities within the same province were sampled during live trapping sessions. Barn Owls preyed mainly upon rodents (79.4% ) and insectivores (18.8%), while birds were eaten to a small degree (1.9%). The main prey taxon was Apodemus sp. (39.7%), followed by Microtus savii (26.1%). The relative frequencies of these two prey items were negatively correlated. Statistically significant differences in the mean prey weight in the thirteen localities were detected. The mean prey weight was positively associated with the wood surface and negatively with the area of cultivated fields. A positive relationship between prey diversity and cultivation was recorded. This indicates that Barn Owls which forage in cultivated habitats tend to prey on smaller species, mainly shrews and voles, and to have a more diversified prey spectrum. On the contrary, owls foraging in wooded areas were more specialized and preyed on larger animals.