Differences in the biological characteristics of the high-risk human papillomavirus type 16 (HPV-16) and the low-risk HPV-6 E7 proteins were analyzed and shown to correlate with certain biochemical properties. To ascertain which region of E7 conferred these properties, chimeric E7 genes were constructed by the exchange of the amino and carboxyl coding halves of the HPV-6 and HPV-16 E7 genes. The amino-terminal half of E7 determined the affinity for binding to the retinoblastoma protein pRB, the transformation properties, and the ability to abrogate transforming growth factor beta-mediated repression of the c-myc promoter. This region of E7 is therefore responsible for the biological and biochemical differences between the E7 proteins of the low-risk and the high-risk HPVs and consequently is one of the critical determinants distinguishing these two groups of viruses. Transcriptional transactivation of the adenovirus E2 promoter, in contrast, was a property shared by E7 proteins of both low-risk and high-risk HPVs.