To determine the function of the E5 open reading frame (ORF) of the human papillomaviruses (HPVs), rodent fibroblast cell lines were transfected with the E5 ORF of HPV type 6 (HPV-6) and HPV-16 expressed from an exogenous promoter. Transfected fibroblasts were transformed to colony formation in soft agar, and the transformation frequency was increased by epidermal growth factor (EGF) but not by platelet-derived growth factor. In a transitory assay, the E5 ORFs from both HPV-6 and HPV-16 were mitogenic in primary human foreskin epithelial cells (keratinocytes) and acted synergistically with EGF. Investigation of keratinocytes expressing HPV-16 E5 showed that the number of endogenous EGF receptors (EGFRs) per cell was increased two- to fivefold. Immunofluorescence microscopy of HPV-16 E5-expressing keratinocytes indicated that there was an apparent delay in the internalization and degradation of EGFRs compared with controls. Kinetic studies with [125I]EGF showed that the ligand underwent normal internalization and degradation in both HPV-16 E5-expressing and control keratinocytes, but in E5-expressing cells, a greater number of receptors recycled back to the cell surface within 1 to 6 h of ligand binding. Finally, ligand-stimulated phosphorylation of the EGFR on tyrosine, an indication of receptor kinase activity, was of greater magnitude in the HPV-16 E5-expressing keratinocytes than in control cells, although the basal level of receptor phosphorylation was similar.