The relationship between bats and coronaviruses (Co. Vs) has received considerable attention since the severe acute respiratory syndrome (SARS)-like CoV was identified in the Chinese horseshoe bat (Rhinolophidae) in 2005. Since then, several bats throughout the world have been shown to shed CoV sequences, and presumably Co. Vs, in the feces; however, no bat Co. Vs have been isolated from nature. Moreover, there are very few bat cell lines or reagents available for investigating CoV replication in bat cells or for isolating bat Co. Vs adapted to specific bat species. Here, we show by molecular clock analysis that alphacoronavirus (ฮฑ-CoV) sequences derived from the North American tricolored bat (Perimyotis subflavus) are predicted to share common ancestry with human CoV (HCoV)-NL63, with the most recent common ancestor between these viruses occurring approximately 563 to 822 years ago. Further, we developed immortalized bat cell lines from the lungs of this bat species to determine if these cells were capable of supporting infection with HCo. Vs. While SARS-CoV, mouse-adapted SARS-CoV (MA15), and chimeric SARS-Co. Vs bearing the spike genes of early human strains replicated inefficiently, HCoV-NL63 replicated for multiple passages in the immortalized lung cells from this bat species. These observations support the hypothesis that human Co. Vs are capable of establishing zoonotic-reverse zoonotic transmission cycles that may allow some Co. Vs to readily circulate and exchange genetic material between strains found in bats and other mammals, including humans. ยฉ 2012, American Society for Microbiology.