Background Understanding the ecological consequences of roads and developing ways to mitigate their negative effects has become an important goal for many conservation biologists. Most mitigation measures are based on road mortality and barrier effects data. However, studying fine-scale individual spatial responses in roaded landscapes may help develop more cohesive road planning strategies for wildlife conservation. Methodology/Principal Findings We investigated how individuals respond in their spatial behavior toward a highway and its traffic intensity by radio-tracking two common species particularly vulnerable to road mortality (barn owl Tyto alba and stone marten Martes foina). We addressed the following questions: 1) how highways affected home-range location and size in the immediate vicinity of these structures, 2) which road-related features influenced habitat selection, 3) what was the role of different road-related features on movement properties, and 4) which characteristics were associated with crossing events and road-kills. The main findings were: 1) if there was available habitat, barn owls and stone martens may not avoid highways and may even include highways within their home-ranges; 2) both species avoided using areas near the highway when traffic was high, but tended to move toward the highway when streams were in close proximity and where verges offered suitable habitat; and 3) barn owls tended to cross above-grade highway sections while stone martens tended to avoid crossing at leveled highway sections. Conclusions Mortality may be the main road-mediated mechanism that affects barn owl and … Palabras clave: Mortality