The human papillomavirus type 16 (HPV16) E5 protein is considered to have weak oncogenic properties, and its function in infected human keratinocytes is unknown. HPV16 E5 protein has been found to localize to the Golgi apparatus and the plasma membrane. To analyze the effect of E5 on plasma membrane properties, cells from the human keratinocyte cell line HaCaT were transfected with the HPV16 E5 open reading frame under the control of an inducible promoter. The gap junction-mediated cell-cell communication of E5- and vector-transfected cells was analyzed by microinjection of Lucifer yellow to measure dye coupling of the cells. A strong impairment of dye transfer in E5-transfected cells but not in vector-transfected cells was observed, with more than 80% dye transfer inhibition 40 min after injection. This impairment correlated with dephosphorylation of connexin 43, the major gap junctional protein in HaCaT cells. Furthermore, the dye coupling inhibition was not the result of differentiation of the E5-expressing cells, since no overexpression of cytokeratin 1 or filaggrin, markers of HaCaT cell differentiation, could be observed. These results therefore strongly suggest a correlation between expression of the HPV16 E5 open reading frame, impairment of gap junction-mediated dye coupling, and dephosphorylation of connexin 43.