BACKGROUND: Many viral pathogens are poorly characterized, are difficult to culture or reagents are lacking for confirmatory diagnoses. We have developed and tested a robust assay for detecting and characterizing large DNA viruses and adenoviruses. The assay is based on the use of degenerate PCR to target a gene common to these viruses, the DNA polymerase, and sequencing the products. RESULTS: We evaluated our method by applying it to fowl adenovirus isolates, catfish herpesvirus isolates, and largemouth bass ranavirus (iridovirus) from cell culture and lymphocystis disease virus (iridovirus) and avian poxvirus from tissue. All viruses with the exception of avian poxvirus produced the expected product. After optimization of extraction procedures, and after designing and applying an additional primer we were able to produce polymerase gene product from the avian poxvirus genome. The sequence data that we obtained demonstrated the simplicity and potential of the method for routine use in characterizing large DNA viruses. The adenovirus samples were demonstrated to represent 2 types of fowl adenovirus, fowl adenovirus 1 and an uncharacterized avian adenovirus most similar to fowl adenovirus 9. The herpesvirus isolate from blue catfish was shown to be similar to channel catfish virus (Ictalurid herpesvirus 1). The case isolate of largemouth bass ranavirus was shown to exactly match the type specimen and both were similar to tiger frog virus and frog virus 3. The lymphocystis disease virus isolate from largemouth bass was shown to be related but distinct from the two previously characterized lymphocystis disease virus isolates suggesting that it may represent a distinct lymphocystis disease virus species. CONCLUSION: The method developed is rapid and broadly applicable to cell culture isolates and infected tissues. Targeting a specific gene for in the large DNA viruses and adenoviruses provide a common reference for grouping the newly identified viruses according to relatedness to sequences of reference viruses and the submission of the sequence data to GenBank will build the database to make the BLAST analysis a valuable resource readily accessible by most diagnostic laboratories. We demonstrated the utility of this assay on viruses that infect fish and birds. These hosts are phylogenetically distant from mammals yet, sequence data suggests that the assay would work equally as well on mammalian counterparts of these groups of viruses. Furthermore, we demonstrated that obtaining genetic information on routine diagnostic samples has great potential for revealing new virus strains and suggesting the presence of new species.