The complete nucleotide sequence of Middelburg virus (MIDV) was determined for strain MIDV-857 from Zimbabwe. The isolation of this virus in 1993 from a horse that died showing severe clinical signs represents the first indication that MIDV can cause severe disease in equids. Full-length cDNA copies of the viral genome were successfully synthesized by an innovative RT-PCR amplification approach using an 'anchor primer' combined with the SMART methodology described previously for the synthesis of full-length cDNA copies from genome segments of dsRNA viruses. The MIDV-857 genome is 11,674 nt, excluding the 5'-terminal cap structure and poly(A) tail (which varies in length from approximately 180 to approximately 220 residues). The organization of the genome is like that of other alphaviruses, including a read-through stop codon between the nsP3 and nsP4 genes. However, phylogenetic analyses of the structural protein amino acid sequences suggested that the MIDV E1 gene was generated by recombination with a Semliki Forest virus-like virus. This hypothesis was supported by bootscanning analysis using a recombination-detection program. The 3' untranslated region of MIDV-857 also contains a 112 nt duplication. This study reports the first full-length sequence of MIDV, which was obtained from a single RT-PCR product.