Human coronavirus (HCV)-229E causes disease only in humans and grows in human cells and in cells of other species that express recombinant human aminopeptidase N (hAPN), the receptor for HCV-229E. We compared the species specificity of HCV-229E infection with the species specificity of virus binding using immunofluorescence, assay of virus yields, fluorescence activated cell sorting and a monoclonal antibody directed against hAPN that blocks infection. We found that HCV-229E binds to intestinal brush border membranes (BBM) and to membranes of cell lines from cats, dogs, pigs, and humans, however the virus only infects two of these species. HCV-229E will not bind to BBM or to membranes from cell lines derived from hamster or mice. Animal coronaviruses related to HCV-229E, including FIPV, CCV, and TGEV bind to cell membranes from cats, dogs, cows, pigs and humans (but not mice), while each virus infects cells from only a subset of these species. Infectious genomic HCV-229E RNA, can infect cells of all of these species. These data suggest that the species-specificity of infection for this serogroup of coronaviruses is determined at the levels of virus binding and penetration. Since binding of viral spike glycoprotein to cellular receptors is not the only limiting factor, we suggest that one or more steps associated with virus penetration may determine the species specificity of infection with the HCV-229E serogroup of coronaviruses.