We evaluated use of 6 wildlife underpasses (UP) using video camera surveillance along State Route 260 in Arizona, USA. We documented wildlife use and compared successful UP crossings by various species and among UP. From 2002 to 2008, we recorded visits by 15,134 animals of 21 species (16 wildlife, 5 domestic) resulting in 72.4% crossing through UP. Elk (Cervus elaphus) accounted for 68% of recorded animals, white-tailed deer (Odocoileus virginianus) and mule deer (O. hemionus) accounted for 13% and 6%, respectively. As elk and white-tailed deer were the only species adequately represented across all UP, we used logistic regression to further evaluate factors associated with successful use of UP. To evaluate habituation over time we limited this analysis to 5 UP monitored for ≥4 yr. For elk, structural attributes and placement, season, time of day, and months monitored were associated with successful elk UP crossing in year 1, however, by year 4 only structural attributes and placement were significant, suggesting that UP structure and placement likely were of primary importance for successful elk passage. By year 4, probabilities of crossing at 4 of 5 UP converged on >0.70, indicating that given sufficient time to allow habituation, most UP we evaluated appeared to be effective for elk, regardless of structural attributes or placement. For deer, only structural attribute and placement were significant, and aside from one structure did not increase in probability of a successful crossing over time. The overall number of animals and species that crossed SR 260 via UP underscores efficacy of UP in promoting multi-species permeability. Long-term monitoring allows wildlife and highway managers to evaluate adaptation to wildlife crossing structures by different species. Results from this study add to our knowledge of mitigating the impact of highways on wildlife. © 2011 The Wildlife Society.