We described previously an African swine fever virus (ASFV) open reading frame, 23-NL, in the African isolate Malawi Lil 20/1 whose product shared significant similarity in a carboxyl-terminal domain with those of a mouse myeloid differentiation primary response gene, MyD116, and the herpes simplex virus neurovirulence-associated gene, ICP34.5 (M. D. Sussman, Z. Lu, G. Kutish, C. L. Afonso, P. Roberts, and D. L. Rock, J. Virol. 66:5586-5589, 1992). The similarity of 23-NL to these genes suggested that this gene may function in some aspect of ASFV virulence and/or host range. Sequence analysis of additional pathogenic viral isolates demonstrates that this gene is highly conserved among diverse ASFV isolates and that the gene product exists in either a long (184 amino acids as in 23-NL) or a short form (70 to 72 amino acids in other examined ASFV isolates). The short form of the gene, NL-S, encodes the complete highly conserved, hydrophilic, carboxyl-terminal domain of 56 amino acids common to 23-NL, MyD116, and ICP34.5. Recombinant NL-S gene deletion mutants and their revertants were constructed from the pathogenic ASFV isolate E70 and an E70 monkey cell culture-adapted virus, MS44, to study gene function. Although deletion of NL-S did not affect viral growth in primary swine macrophages or Vero cell cultures in vitro, the null mutant, E70/43, exhibited a marked reduction in pig virulence. In contrast to revertant or parental E70 where mortality was 100%, all E70/43-infected animals survived infection. With the exception of a transient fever response, E70/43-infected animals remained clinically normal and exhibited a 1,000-fold reduction in both mean and maximum viremia titers. All convalescent E70/43-infected animals survived infection when challenged with parental E70 at 30 days postinfection. These data indicate that the highly conserved NL-S gene of ASFV, while nonessential for growth in swine macrophages in vitro, is a significant viral virulence factor and may function as a host range gene.