Infection with mucosal high risk Human Papilloma Viruses (HPVs) is associated with increased risk of cervical neoplasia and cancer. The HPV replicative life cycle is highly dependent on the differentiation process of the infected epithelia, and HPV gene expression is therefore tightly regulated. We could demonstrate for the first time that alternative splicing of HPV16 E6/E7 open-reading frame cassette is regulated by the epidermal growth factor (EGF) pathway. The presence of EGF was coupled to preferential E6 expression, while depletion of EGF, treatment with EGF-receptor (EGFR) neutralizing antibodies or with the EGFR-inhibitor tyrphostin AG1478 resulted in E6 exon exclusion in favor of E6*. As a consequence, increased p53 levels and enhanced translation of E7 with a subsequent reduction of the retinoblastoma protein pRb could be discerned. E6 exon exclusion upon EGF depletion was independent from promoter usage, mRNA stability or selective mRNA transport. Time-course experiments and incubation with cycloheximide demonstrated that E6 alternative splicing is a direct and reversible effect of EGF signal transduction, not depending on de novo protein synthesis. Within this process, Erk1/2-kinase activation was the critical event for E6 exon inclusion, mediated by the upstream MAP kinase MEK1/2. Moreover, siRNA knockdown experiments revealed an involvement of splicing factors hnRNPA1 and hnRNPA2 in E6 exon exclusion, whereas the splicing factors Brm and Sam68 were found to promote E6 exon inclusion. Since EGF and EGFR expression as well as EGFR signalling is known to be modulated during differentiation and transformation, it is tempting to speculate about the relevance of EGF-modulated E6/E7 expression for both, the viral life cycle and the HPV-induced carcinogenesis process.