Human papillomavirus (HPV) type 16 belongs to the group of "high risk" HPV types that are frequently detected in anogenital cancers. The expression of HPV-16 late genes encoding the virus capsid proteins L1 and L2 is restricted to terminally differentiated epithelial cells in the superficial layers of the squamous epithelium. We have previously identified negative elements in the 3' end of L2 RNA that act in cis to reduce mRNA utilization without substantially affecting mRNA levels. The experiments reported here demonstrate the interaction of cellular proteins with an inhibitory sequence present in the coding region of the L2 mRNA. Using RNA gel shift assays and UV cross-linking, we have detected three cellular proteins interacting specifically with the sense strand of the L2 mRNA, two of which were identified as heterogeneous ribonucleoprotein K (hnRNP K) and the poly(rC) binding- protein (PCBP). Recombinant hnRNP K, PCBP-1, and PCBP-2 that were over expressed in bacteria and partially purified bound to the HPV-16 L2 mRNA in a sequence-specific manner. Interestingly, PCBP-1, PCBP-2, and hnRNP K specifically and efficiently inhibited translation of the HPV-16 L2 mRNA in vitro. Therefore, these proteins may play an important role in the regulation of HPV-16 late gene expression and virus production in vivo.