Human papillomavirus type-16 (HPV-16) transcription in two human keratinocyte cell lines (HPK) immortalized by transfection of viral DNA in vitro was analyzed by nucleotide sequencing of cDNA clones, and in addition by primer extension analysis and S1 nuclease and exonuclease VII digestion of poly(A)+ RNA. A novel mRNA species which probably initiates in the E7 ORF and in which the 5'-part of the E1 ORF (splice donor at position (pos.) 880) is joined to an exon comprising the entire E2 ORF (splice acceptor at pos. 2708) was found in both cell lines. This mRNA has the potential to encode a full-length E2 protein, which is known to function as a repressor of transcription initiated at P97. cDNAs derived from the late region of the viral genome and the use of a late polyadenylation signal at pos. 7320-7325 are described. In agreement with RNA data published by others the major promoter for HPV-16 transcription is located at pos. 97. mRNA species encoding full-length or truncated forms of the E6 protein, and species characterized by an E1i [symbol see text] E4 splice junction (which provided the E4 open reading frame (ORF) with an ATG triplet) were identified.