When bovine papillomavirus transforms cells in vitro, it maintains its genome as a multicopy nuclear plasmid. Plasmid DNA extracted from such transformed cells was analyzed by the two-dimensional gel electrophoresis technique of Brewer and Fangman (B. Brewer and W. Fangman, Cell 51:463-471, 1987). The replication intermediates detected in these assays were found to be the sums of the oligomeric and monomeric forms of the replicating plasmids. The multimeric DNAs were shown by field inversion gel electrophoresis and partial restriction digestion to be head-to-tail concatemers of the monomeric forms. Furthermore, the multimers progressed in size by steps of one monomer, indicating that they did not arise by replication segregation mistakes of the unit length, which would predict a ladder spaced by integrals of two monomers. To map the plasmid DNA replication origin, the replication intermediates of the monomers were isolated by successive sucrose gradient centrifugation and then examined by the two-dimensional gel electrophoresis method. The patterns detected show that bovine papillomavirus type 1 replicates in these cells bidirectionally and that one replication origin site in the viral genome is utilized. By employing several restriction enzymes and specific viral DNA probes to dissect the replication intermediates, we were able to map the origin of initiation site with some precision. The initiation site, which maps to bovine papillomavirus type 1 DNA position 7730 +/- 100 bp, places the origin within that region of the viral upstream regulatory region which contains the major cluster of transcription factor E2-binding sites, E2RE1. Thus, the actual viral plasmid origin of replication maps near, but outside, genetic elements previously shown to be important for plasmid maintenance.