The principal molecular determinant of virulence of Newcastle disease virus (NDV) is the amino acid sequence at the fusion cleavage activation site. To extend the understanding of the role of the fusion cleavage activation site in NDV virulence, the pathogenesis in chickens of a lentogenic LaSota isolate and two infectious clones, NDFL and NDFLtag, were compared. NDFL is an infectious clone of a lentogenic NDV strain (LaSota E13-1), and NDFLtag is the infectious clone with the fusion cleavage site sequence mutated to the virulent motif. NDFL and NDFLtag were described by Peeters et al. The viruses were inoculated intraconjunctivally into groups of 4-wk-old white leghorn chickens and compared in a pathogenesis study for determination of disease causation (clinical signs of disease, gross lesions, histology, virus isolation, and serology) and viral distribution (presence of viral nucleoprotein and mRNA was detected by immunohistochemistry and in situ hybridization, respectively). The modification of the fusion cleavage activation site to the virulent motif in the infectious clone only slightly increased disease severity and viral distribution in the pathogenesis assessment, even though dramatically increased pathogenicity of NDFLtag was confirmed by standard pathogenicity index tests. The result, that the mutated fusion cleavage site of NDV–NDFLtag had only a small influence on pathogenesis in chickens compared to either E13-1 or NDFL, suggests that the pathogenic effects of NDV are not dependent on the fusion cleavage site alone.