Lack of permissive and productive cell cultures for the human papillomaviruses (HPVs) has hindered the study of virus-neutralizing antibodies and infection. We developed a cell-free system generating infectious HPV16 pseudovirions. HPV16 L1/L2 capsids, which had been self-assembled in insect cells (Sf9) expressing virion proteins L1 and L2, were disassembled with 2-mercaptoethanol (2-ME), a reducing agent, and reassembled by removal of 2-ME in the presence of a β-galactosidase expression plasmid. Plasmid DNA purified together with the reassembled capsids was resistant to DNase I digestion. The reassembled pseudovirions mediated DNA transfer to COS-1 cells, as monitored by induced β-galactosidase activity. Transfer was inhibited by anti-HPV16 L1 antiserum but not by antisera against L1s of HPV6 and HPV18. Construction in vitro of HPV pseudovirions containing marker plasmids would be potentially useful in developing methods to assay virus-neutralizing antibodies and to transfer exogenous genes to HPV-susceptible cells.