Expression of the betapapillomavirus (betaPV) E6/E7 genes has been shown to impair both keratinocyte differentiation and apoptosis. Especially late-terminal keratinocyte differentiation shares certain aspects with apoptosis, such as fragmentation of DNA and activation of caspases. Here we investigated the disruption of keratinocyte differentiation in organotypic skin (raft) cultures of primary (PHK) and immortalized (N/TERT) human keratinocytes, in particular by human papillomavirus (HPV)8.Immunohistochemical analysis of HPV5 and HPV8 E6/E7-expressing PHK revealed thickening of the rafts and complete absence of stratum corneum formation, even after 18 days of culture. This phenotype was confirmed in N/TERT raft cultures. When expressed separately, the aberrant morphology was observed only in rafts expressing E6, not E7. Immunofluorescence analysis of HPV8 E6 PHK rafts showed an increase in number and size of Filaggrin- and Caspase-14-positive cells in the granular layer. In raft lysates analyzed by western-blot, the presence of pro-Caspase-14 in the differentiated keratinocytes was confirmed, but in the HPV8 E6 rafts none of the Caspase-14 subunits were detected.In conclusion, in the raft system, HPV8 E6 prevented late-terminal keratinocyte differentiation resulting in an accumulation of Filaggrin and pro-Caspase-14-positive cells in the absence of stratification. This differentiation arrest was accompanied by the failure to express Caspase-14 subunits, suggesting absence of Caspase-14 activation and probable abrogation of Filaggrin maturation in HPV8 E6-expressing keratinocytes.