Two pathogenetically different pigeon paramyxovirus type 1 (PPMV-1) virus clones were recently derived by passage of a single isolate with an intracerebral pathogenicity index (ICPI) of 0.32. The virus clones had an ICPI of 0.025 and 1.3, respectively (Fuller et al., 2007). Remarkably both viruses contained a cleavage site motif in the precursor fusion (F) protein that is usually associated with virulent viruses. In the current study, both viral genomes were completely sequenced and only four amino acid differences were observed. Of these, two were considered irrelevant on theoretical grounds and two amino acid changes were unique for virus 0.025. The latter were introduced into an infectious clone of a virulent Newcastle disease virus strain, individually and combined, and the effects of the mutations on pathogenicity were examined. The results indicate that only the S453P substitution in the F protein had a modest effect on pathogenicity. We were not able to identify the molecular basis for the pathogenicity difference between both viruses. However, our observations emphasize the need to determine both the virulence (ICPI) and the sequence of the cleavage site of the F protein to avoid dismissing of potential virulent PPMV-1 isolates.