Human papillomavirus type 16 (HPV-16) is strongly associated with the development of cervical cancer. Studies of model systems with animal papillomaviruses have demonstrated the importance of neutralizing antibodies in preventing papillomavirus-associated disease. The assessment of neutralizing antibody responses against HPV-16, previously hampered by the lack of a viral source, was enabled by the recent propagation of an HPV-16 stock in xenografted severe combined immunodeficiency (SCID) mice. HPV-16 infection of an immortalized human keratinocyte cell line was demonstrated by detection of an HPV-16-specific spliced mRNA amplified by reverse transcriptase PCR. Infection was blocked by preincubation of the virus with antiserum generated against HPV-16 virus-like particles (VLPs) composed of the major capsid protein, L1. To examine potential cross-neutralizing activity among the different genital HPV types, rabbit antisera to L1 VLPs corresponding to HPV-6, -11, -18, -31, -33, -35, -39, and -45 were assayed for the ability to block the HPV-16 infection of cultured cells. Antiserum raised against HPV-33 L1 VLPs was the only heterologous antiserum which inhibited HPV-16 infection. Thus, a neutralization assay for HPV-16 may help to characterize the components required to compose a broadly efficacious genital HPV vaccine.