A PCR protocol was established that not only allows the detection of, but also the differentiation of species of the genus Orthopoxvirus. This assay was accomplished by the selection of oligonucleotides located within the gene that encodes the A-type inclusion protein of cowpox virus. The primer pair flanked a region exhibiting distinct and specific DNA deletions in the corresponding sequences of vaccinia, mousepox, monkeypox and camelpox virus. For this reason, PCR resulted in DNA fragments of different sizes. The presented PCR protocol, combined with BglII restriction digests, allowed the unequivocal assignment of 42 orthopoxvirus (OPV) strains and isolates to the correct OPV species. The resulting classification corresponded exactly with known biological data for the OPV strains investigated. Furthermore, 13 out of 22 cowpox virus isolates could be subtyped by the presence or absence of a small BglII fragment. DNA sequencing showed that the lack of this BglII fragment was caused by a deletion of 72 nucleotides.