The ligase chain reaction was used to assess the virulence of isolates of Newcastle disease virus. In the main study, 18/18 virulent isolates whose nucleotide sequences that code for the cleavage site and fusor peptide regions were known, successfully ligated oligonucleotides in a primer mix for virulent viruses termed VPM. Five of these isolates yielded a more intense ligated product with a second primer mix for virulent viruses called VPM1. No ligation was evident with eight avirulent isolates in tests with VPM or VPM1, however, each of these viruses did yield a strong ligated product with the primer mix for avirulent viruses (AVPM) as did one virulent isolate considered to be a mixture. Two virulent Australian isolates, 1238/1998 and 1248/1998, showed low but seemingly specific ligation with AVPM. In a blind study, 8/9 virulent isolates whose sequences were unknown ligated primers in VPM. Three avirulent and one virulent isolate, the latter again probably a mixture, ligated primers in AVPM. Ligation of oligonucleotides in VPM and AVPM was detectable in mixtures where virulent and avirulent isolates represented 0.1% and 0.01% by volume respectively of the viral population. The results indicate that LCR offers a potential in vitro alternative to current in vivo tests for virulence determination of Newcastle disease virus isolates.