Twenty-two infectious bursal disease virus (IBDV) strains were examined using the reverse transcriptase/polymerase chain reaction-restriction endonuclease (RT/PCR-RE) assay. A 394-bp fragment of the VP2 gene was amplified and tested for six different restriction enzyme sites. Although the EcoRII enzyme was used in previous RT/PCR-RE assays, results obtained using the isoschizomer BstNI were more consistent because its activity does not rely on multiple restriction sites. Ten different RT/PCR-RE profiles were observed. IBDV strains previously reported to be variant type viruses were BstNI and StyI negative except variant strain IN. The Bursine, Bursine-2, Bursine-Plus and Bio-Burs viruses were BstNI negative. The presence of a second enzyme site, StyI, was observed in these viruses and could be used to differentiate them from the known variant viruses, which were StyI negative. Nucleotide sequence data also indicated that these viruses were not identical to variant or classic type viruses. The base substitution observed in the BstNI site of Bio-Burs and Bursine-2 was responsible for changing the amino acid at position 222 to serine. The amino acid at this position has been reported to influence a neutralizing epitope on VP2. Three IBDV strains were examined after propagation in different hosts. The RE profiles of the STC and MD IBDV strains did not change after propagation in either BGM-70 cell culture or chicken bursas, whereas the Del-A RE profile changed at the Sau3AI site after adaptation to BGM-70 cell culture. This site has not been associated with antigenic or other phenotypic characteristics of IBDV.