The cis and trans signals important in BPV replication were identified using a transient replication assay, the mobility shift assay, and a comparison between the BPV and LPV genomes. Replication of deleted BPV genomic clones, which contain the natural left (3’ OH end of the viral minus strand) and right (5’ PO, end of the viral minus strand) BPV termini, defined the minimum size of the BPV origin of replication (ori) to be the terminal 171 nucleotides of each terminus. Clones containing duplicate termini or altered left ends were also shown to replicate. The BPV ori was determined to have two domains identified by a computer analysis of homologus regions between these termini. Three proteins were identified that bind to the left terminal 171 nucleotides in the hairpin conformation. Inhibition of the formation of the DNA-protein complexes with competitor DNA localized two potential binding sites that correspond to the domains mentioned above. Two of the DNA-protein complexes were formed by BPV-coded proteins as determined by inhibition of the complex by anti-BPV antibodies. The third complex resulted from binding of a host cell S-phase protein that is a likely candidate for the S-phase factor required for autonomous parvovirus replication. The BPV ori thus appears to function by binding both cellular and viral proteins for the initiation of DNA synthesis from the hairpinned termini. The comparison of the BPV and LPV genome sequence suggest that the genomic organization of LPV may be more like BPV than that of the rodent parvovirus minute virus of mice; and therefore, LPV may contain similar cis signals.