The genomic replicative form DNAs (RF-DNA) of three maize streak virus isolates (MSV-CT, MSV-PE, and MSV-SW) from widely separated locations in southern Africa were characterized by restriction endonuclease mapping in order to assess the feasibility of using the technique to determine genetic variability between isolates. The viruses were transmitted to and propagated in laboratory-grown maize by the leafhopper vector Cicadulina mbila (Naudé). MSV-PE produced more severe symptoms than MSV-CT and MSV-SW; the isolates were serologically identical in 'western' immunoblot tests, but distinct in 'sandwich' enzyme-linked immunosorbent assays. RF-DNA of all three isolates was prepared from infected maize; the RF-DNA of MSV-CT and MSV-PE was cloned in a plasmid vector in Escherichia coli. Restriction maps were generated from this cloned DNA and from the RF-DNA of MSV-SWA. The maps were similar in regions expected to be conserved, but there were also important differences between all isolates. The implications of these results, and of relationships amongst these and other sequenced isolates of MSV, are discussed.