Human papillomavirus (HPV) type 1 (HPV 1) is associated with benign cutaneous warts and HPV type 16 (HPV 16) with mucosal epithelial lesions that can progress to invasive carcinoma. The primary structure of the HPV E4 proteins is not highly conserved between types and their role in the viral life cycle is still unknown. A large panel of Simian virus 40 (SV40)-transformed human and monkey epithelial and fibroblast cell lines were infected with recombinant SV40/HPV1 E4 or SV40/HPV 16 E4 viruses and the expression of the viral proteins was analyzed by indirect immunofluorescence. Both HPV 1 and HPV 16 E4 proteins formed extensive and organized filamentous cytoplasmic networks that co-localized with the cytokeratin intermediate filaments. However, only HPV 16 E4 induced the collapse of the cytokeratin filaments. Furthermore, when both virus type E4 proteins were expressed within the same cell the collapse of the HPV 16 E4 filaments did not induced the collapse of the HPV 1 E4 network. Similar E4 filamentous structures were also observed in the cytoplasm of cells of the parabasal layer of an HPV 1-induced experimental wart. The HPV 16 E4 protein formed cytoplasmic networks in all SV40-transformed cell lines examined, but HPV 1 E4 only formed filamentous networks in human keratinocytes and in a monkey stomach epithelial cell line. In keratinocyte cells HPV 1 E4 species of 16, 17, 32, and 34 kDa were expressed, while in Cos-1 cells (in which no E4 networks are formed) only the 17 and 34 kDa polypeptides were found. The specific behavior of E4 proteins of cutaneous and mucosal HPVs expressed in cultured cells may suggest that these viral proteins have evolved to perform a similar function at different epithelial sites.