The molecularly cloned, prototype human papillomavirus (HPV) 16 DNA has a single-base deletion in the El gene. We reconstructed a putative non-defective HPV 16 genome with an uninterrupted El gene, and examined its biological functions. The reconstructed HPV 16 DNA formed foci of morphologically transformed cells in transfected rat 3Y1 cell cultures (an immortalized normal cell line). The transformed rat cells contained transcriptionally active HPV 16 DNA, which appeared to be integrated within the cell DNA, and formed colonies in soft agar.