The internalization pathway and mechanism of uncoating of human rhinovirus serotype 2 (HRV2), a minor-group human rhinovirus, were investigated. Kinetic analysis revealed a late endosomal compartment as the site of capsid modification from D to C antigenicity. The conformational change as well as the infection was prevented by the specific V-ATPase inhibitor bafilomycin A1. A requirement for ATP was also demonstrated with purified endosomes in vitro. Capsid modifications occurred at a pH of 5.5 regardless of whether the virus was entrapped in isolated endosomes or free in solution. These findings suggest that the receptor is not directly involved in the structural modification of HRV2. Viral particles found in purified endosomes of infected cells were mostly devoid of RNA. This supports the hypothesis that uncoating of HRV2 occurs in intact endosomes rather than by a mechanism involving endosomal disruption with subsequent release of the RNA into the cytoplasm.