To study the role of human papillomavirus (HPV) infection in the development of genetic instability, we transduced normal human airway and anogenital epithelial cells with various combinations of HPV-16 E6, E7, and the reverse transcriptase component of telomerase (hTERT). Cell lines generated by co-expression of E7 with E6 and/or hTERT (i.e., E6/E7, E7/hTERT, and E6/E7/hTERT) exhibited extra copies of chromosome 20 and specific amplification of the 20q12-ter region, whereas those generated without E7 (i.e., hTERT alone or E6/hTERT) did not. Co-expression of hTERT and a dominant-negative version of cdk4 that has been shown to inactivate the retinoblastoma (pRb) pathway also resulted in 20q amplification. Interestingly, extra copies of chromosome 20 were observed in early passage keratinocytes that expressed E7 alone, and microarray expression analysis revealed that genes in the 20q region and on chromosome 5 were specifically upregulated in these cells. Our results indicate that chromosome 20q amplification is an early event that may be specifically caused by expression of E7 through inactivation of the pRb pathway in human epithelial cells.