In this study we have examined the pathway by which papillomaviruses infect cells, using bovine papillomavirus (BPV) virions and mouse C127 cells as the model system. By confocal microscopy, the entry of BPV virions, BPV virus-like particles (VLPs), and HPV16 VLPs were very similar. In dually exposed cells, HPV-16 VLPs and BPV virions colocalized intracellularly. BPV VLPs colocalized with AP-2, a clathrin adapter molecular and a marker of the clathrin-dependent endocytic pathway; and also with transferrin receptor, a marker of early endosomes; and Lamp-2, a marker of late endosomes and lysosomes. BPV infection was detected within 12 h of virion cell-surface binding, as measured by an RT-PCR assay. Infection was prevented by several pharmacologic inhibitors, including chlorpromazine, which blocks clathrin-dependent endocytosis and the lysosomotropic agent, bafilomycin A. By contrast, two inhibitors of caveolae-dependent uptake, filipin and nystatin, did not prevent BPV infection. We conclude that papillomaviruses infect cells via clathrin-dependent receptor-mediated endocytosis. Surprisingly, the kinetics of internalization were unusually slow for this mechanism, with the t(1/2) of entry of BPV-1 being approximately 4 h versus 5-15 min for a typical ligand.