Foot-and-mouth disease (FMD) virus causes a highly contagious, economically important disease of cloven-hoofed animals, including livestock animals such as cattle and swine. In South Africa, the disease is controlled primarily through prophylactic vaccination and strict animal movement control. To control effectively the spread of FMD, it has become increasingly more important to develop diagnostic tests that can differentiate FMDV-infected animals from those that have been vaccinated. Currently, the detection of antibodies to nonstructural proteins, especially the 3ABC nonstructural polypeptide, is considered to be the most reliable method to distinguish virus-infected from vaccinated animals. Towards the development of such a serological test, the primary aim of this investigation was to express the 3ABC nonstructural polypeptide of the SAT2 type FMD virus ZIM/7/83 in both a prokaryotic and eukaryotic expression system in order for the recombinant protein to be used as a diagnostic antigen. The nucleotide sequence of the 3ABC-encoding region of SAT2/ZIM/7/83 was determined, the amino acid sequence deduced and subsequently compared to corresponding sequences of other virus isolates representing all seven FMDV serotypes. Phylogenetic analysis revealed that the 3ABC polypeptide of the SAT serotypes, which are mostly restricted to sub-Saharan Africa, clustered separately from the euroasiatic FMDV serotypes (types A, O, C and Asia1). Amino acid sequence alignments also indicated considerable variation in the 3A, 3B and 3C proteins between the SAT and euroasiatic types located mainly in previously identified epitope-containing regions. These results suggest that the sensitivity and specificity of diagnostic tests based on the 3ABC nonstructural polypeptide of the European FMDV types may be compromised when applied to the African sub-continent. Therefore, a SAT-specific diagnostic assay is required to distinguish virus-infected from vaccinated animals. The 3ABC-encoding region of SAT2/ZIM/7/83 was subsequently expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein using the bacterial expression vector pGEX-2T, and in Spodoptera frugiperda insect cells using the BAC-to-BACTM baculovirus expression system. Although high-level expression of the recombinant GST-3ABC protein was obtained, the GST-3ABC protein was insoluble and could not be purified by glutathione affinity chromatography. Therefore, the recombinant GST-3ABC fusion protein was purified from reverse-stained SDS-polyacrylamide gels and shown to be immunoreactive in Western blot analysis using an FMDV-specific serum. Expression of the 3ABC polypeptide in insect cells infected with a recombinant bacmid yielded soluble recombinant protein, but the level of expression was lower compared to that obtained in E. coli. In addition, Western blot analysis of cell extracts prepared from recombinant bacmid-infected cells revealed the presence of three immunoreactive proteins of 47, 25 and 18 kDa. These correspond with the size of the FMDV proteins 3ABC, 3AB and 3A, respectively, suggesting that the 3C protease was responsible for proteolytic cleavage of the 3ABC polypeptide. Based on the results obtained, the bacmid expression system appears to be more suitable for the production of the 3ABC polypeptide.