The human papillomavirus (HPV) life cycle is linked to the differentiation state of the host cell. In virus-infected undifferentiated basal epithelial cells, HPV genomes are maintained as episomes at low copy number. Upon differentiation, a concomitant increase in viral copy number and an induction of late gene expression from a differentiation-specific promoter is seen. To investigate whether late gene expression was dependent on the amplification of the viral genome, inhibitors of DNA replication and in vitro systems for epithelial differentiation were used in conjunction with cells that stably maintain HPV31 episomes. Treatment of cells induced to differentiate in methylcellulose with the DNA synthesis inhibitor cytosine beta-arabinofuranoside (AraC) blocked viral DNA amplification but did not prevent induction of late transcription. This suggests that late gene expression does not strictly require amplification of the viral genome and that differentiation signals alone are sufficient to activate transcription from the late promoter. However, DNA amplification does appear to be necessary for maximal induction of the late promoter. In order to examine the cis-acting elements that contribute to the activation of the late promoter, a transient reporter assay was developed. In these assays, an induction of late gene expression was seen upon differentiation that was specific to the late promoter. Mapping studies localized important regulatory elements to the E6/E7 region and identified short sequences that could serve as binding sites for transcription factors. Elements within the upstream regulatory region were also found to positively and negatively influence transcription from the late promoter. These results identify mechanisms important for the differentiation-dependent activation of late gene expression of high-risk papillomaviruses.