Human papillomaviruses (HPVs) are aetiological agents of human malignancies, most notably cervical cancers. The life-cycles of HPVs are dependent on epithelial differentiation, and this has impeded many basic studies of HPV biology. The organotypic (raft) culture system supports epithelial differentiation such that infectious virions are synthesized in raft tissues from epithelial cells that replicate extrachromosomal HPV genomes. The CIN-612 9E cell line maintains episomal copies of HPV type 31b (HPV31b), an HPV type associated with cervical cancers. Many previous studies, including our own, have focused on characterizing the later stages of the HPV31b life-cycle in CIN-612 9E raft tissues. In this study, we have used the raft system to generate large numbers of HPV31b viral DNA (vDNA)-containing particles. We found a biologically contained homogenization system to be efficient at virion extraction from raft epithelial tissues. We also determined that vDNA-containing particles could be directly quantified from density-gradient fractions. Using an RT-PCR assay, the presence of newly synthesized, spliced HPV31b transcripts was detected following HPV31b infection of the immortalized HaCaT epithelial cell line. Spliced E6 and E1( wedge )E4 RNAs were detected using a single round of RT-PCR from cells infected with a dose as low as 1.0 vDNA-containing particle per cell. Spliced E1*I,E2 transcripts were found in cells infected with an HPV31b dose as low as 10 vDNA-containing particles per cell. Infectivity was blocked by HPV31 antiserum, but was not affected by DNase I. This work lays a foundation for a detailed analysis of the early events in HPV infection.