The papovaviridae family consists of two genera, the papillomaviruses (PV) and the polyomaviruses (Py-V). Both genera are distinguished by morphological (larger sizes of the PV) and several biological characteristics. The genomes of either of the two genera share highly conserved DNA regions and a common antigenic determinant, located in their major capsid polypeptides. On the basis of these data an evolutionary relationship among the members of PV and Py-V, respectively, has been suggested. No homology has been found for either DNA- or protein sequences between PV and Py-V and the question of a common ancestor for both viral genera remains open. We have started to characterize the genome of a papilloma producing papovavirus of the Syrian hamster (HaPV). Most of the known biological characteristics of the HaPV suggest it should be classified as a papilloma-like virus. However, the molecular weight of about 3.5 X 10(6) daltons found for the circular duplex DNA lies within the range given for SV 40 and polyoma virus (Py). Analysis of the HaPV genome by cleavage with 21 different restriction endonucleases, location of specific binding sites of phage T 4 gene 32 protein and E. coli RNA polymerase on the viral DNA demonstrated that the HaPV differed distinctly from all other currently known papovaviruses. The HaPV genome was also analyzed by filter hybridization and electron microscopy under conditions of varied stringency for nucleotide sequence homology with the genomes of different papovaviruses of both genera. Whereas no homologous DNA regions could be found between the genomes of HaPV and the human PV types 1 and 4, only under nonstringent conditions (Tm-43 degrees C) stable hybrids were formed between HaPV-, SV 40- and the DNA of a PV isolated from Mastomys natalensis (MnPV). On the other hand extensive homology was detected between the genomes of HaPV and Py even under stringent hybridization conditions (Tm-28 degrees C). The homologous DNA segments mapped on the Py and partially on the SV 40 genome were found to be the most strongly conserved DNA regions among the Py-V genus. These results are discussed with respect to a classification of the HaPV within the papovaviridae family.