The central variable region (CVR) of the African swine fever virus (ASFV) genome is contained within the 9-RL open reading frame (ORF). ORF 9-RL of the ASFV isolate Malawi Lil-20/1 predicts a protein of 614 amino acids with amino- and carboxy-terminal hydrophobic regions and a centrally located hydrophilic region. The CVR of the genome, located centrally within this ORF, is 372 bp and contains a 132-bp direct repeat. The translated CVR within ORF 9-RL contains 31 tandem tetramers, predominantly NADT, NANT, NVDT, and, in a few cases, CAST, GAST, or CADT. In vitro translation of 9-RL yielded a 94-kDa protein that was strongly reactive with convalescent pig serum while monospecific 9-RL antiserum identified a late viral protein of 94 kDa in ASFV-infected macrophages. The protein, detected by immunofluorescence staining with 9-RL antiserum, was distributed homogeneously throughout the cytoplasm of infected Vero cells. 9-RL protein size varied among different viral isolates and among cell-culture-adapted viruses. Protein size increased proportionately with the degree of cell culture adaptation and was directly correlated with the size of the CVR present within the ORF (300-500 bp). Analysis of the number and composition of tandem tetramers present within the CVR of a given ASFV isolate may prove useful for identifying and/or grouping ASFV isolates.