During a Myotis sodalis telemetry project in Pennsylvania, USA, in 2000, road-killed M. lucifugus were recorded and a highway survey was initiated. The purpose of this study was to assess the level of mortality from road kills on this colony, verify which species were being killed in traffic and examine the influence of canopy height and structure on flight behavior. On 10 evenings between 15 May and 26 July 2001, bats were counted as they emerged from day roosts and crossed a heavily trafficked highway en route to foraging areas. A total of 26 442 bats were observed cross- ing this highway over 9.29 h of observation. Bats used canopy cover when approaching the highway from roosts. Where canopy cover was lacking adjacent to the highway, fewer bats were counted crossing; where adjacent canopy was low (≤6 m), bats crossed lower and closer to traffic. Motivated by a planned highway upgrade that would extend deforestation of the highway verge, we monitored the flight behavior of M. lucifugus as they crossed a 55 m mowed field. Observations of more than 1700 bats revealed that a vast majority of commuting individuals fly less than 2 m above the ground in the open field. Between 15 May and 14 September 2001, searches for road-killed bats were con- ducted along a section of highway crossed by large numbers of bats. We collected 27 road-killed M. lucifugus, 1 suspected M. sodalis and 1 unidentifiable Myotis sp. Subsequent genetic analyses of mitochondrial DNA sequences confirmed the suspect bat as a federally endangered M. sodalis.