The African swine fever virus (ASFV) genome contains a gene, 9GL, with similarity to yeast ERV1 and ALR genes. ERV1 has been shown to function in oxidative phosphorylation and in cell growth, while ALR has hepatotrophic activity. 9GL encodes a protein of 119 amino acids and was highly conserved at both nucleotide and amino acid levels among all ASFV field isolates examined. Monospecific rabbit polyclonal antibody produced to a glutathione S-transferase-9GL fusion protein specifically immunoprecipitated a 14-kDa protein from macrophage cell cultures infected with the ASFV isolate Malawi Lil-20/1 (MAL). Time course analysis and viral DNA synthesis inhibitor experiments indicated that p14 was a late viral protein. A 9GL gene deletion mutant of MAL (9GL), exhibited a growth defect in macrophages of approximately 2 log₁₀ units and had a small-plaque phenotype compared to either a revertant (9GL-R) or the parental virus. 9GL affected normal virion maturation; virions containing acentric nucleoid structures comprised 90 to 99% of all virions observed in 9GL-infected macrophages. The 9GL virus was markedly attenuated in swine. In contrast to 9GL-R infection, where mortality was 100%, all 9GL-infected animals survived infection. With the exception of a transient fever response in some animals, 9GL-infected animals remained clinically normal and exhibited significant 100- to 10,000-fold reductions in viremia titers. All pigs previously infected with 9GL survived infection when subsequently challenged with a lethal dose of virulent parental MAL. Thus, ASFV 9GL gene deletion mutants may prove useful as live-attenuated ASF vaccines.