The complete nucleotide sequence of the doubt-stranded circular DNA of bovine papillomavirus type 1 (BPV-1) was determined. Analysis of this sequence in conjunction with known transcriptional data for the virus provides a basis of determining the organization of the papillomavirus genome. All the major open reading frames are located on the same DNA strand. The region transcribed in BPV-transformed cells contains open frames in all three translational frames whereas the region transcribed in productively infected bovine fibropapillomas is characterized by two large open reading frames partitioned by a single translational stop codon. The localization of sequences diagnostic of promoters and polyadenylation sites suggests that splicing is involved in the biosynthesis of the viral mRNAs. A sequence comparison indicates the genome organization of the bovine papillomavirus and that of the members of the simian virus 40-polyomavirus subgroup to be distinct, suggesting that these two groups of viruses are evolutionarily unrelated.