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Abstract Pathogens with persistent environmental stages can have devastating effects on wildlife communities. White-nose syndrome (WNS), caused by the fungusPseudogymnoascus destructans,has caused widespread declines in bat populations of North America. In 2009, during the early stages of the WNS investigation and before molecular techniques had been developed to readily detectP. destructansin environmental samples, we initiated this study to assess whetherP. destructanscan persist in the hibernaculum environment in the absence of its conclusive bat host and cause infections in naive bats. We transferred little brown bats (Myotis lucifugus) from an unaffected winter colony in northwest Wisconsin to twoP. destructanscontaminated hibernacula in Vermont where native bats had been excluded.Infection withP. destructanswas apparent on some bats within 8 weeks following the introduction of unexposed bats to these environments, and mortality from WNS was confirmed by histopathology at both sites 14 weeks following introduction. These results indicate that environmental exposure toP. destructansis sufficient to cause the infection and mortality associated with WNS in naive bats, which increases the probability of winter colony extirpation and complicates conservation efforts.