The host's immune reaction against human papillomavirus (HPV) infection remains poorly understood. Inflammatory cytokines undoubtedly play a key role through activating and coordinating the immune response. However, their direct interactions with the HPV genes remain unclear. In the present study, the effects of various inflammatory cytokines on HPV16 gene expression were investigated. In a CAT assay, tumor necrosis factor (TNF) alpha and interleukin-1 (IL-1) alpha were shown to repress HPV16 early gene expression at the transcriptional level through the noncoding region (NCR), whereas IL-6 and interferon-gamma did not. In Northern blot analysis, TNF and IL-1 were also shown to repress HPV16 E6/E7 mRNA expression in the HPV16-immortalized human keratinocyte cell line. The TNF- and IL-1-responsive elements in the HPV16 NCR were determined to lie within the cell-type-specific enhancer, where there are several binding sites for nuclear factors involved in HPV16 early gene regulation, suggesting the participation of these factors in TNF and IL-1 regulations. Thus, TNF and IL-1 were shown to have antiviral effects on HPV through down-regulation of its gene transcription. This is the first demonstration that TNF and IL-1 are involved in HPV gene regulation. These functions of inflammatory cytokines are presumed to contribute to the host's defense against HPV infection.