An outbreak of rabies in a pack of endangered wild dogs (Lycaon pictus) in the Madikwe Game Reserve, South Africa, is described. The outbreak, which occurred in early 2000, was the second outbreak of rabies in wild dogs followed their re-introduction to the Reserve in 1995. The outbreak resulted in the death or disappearance of 10 out of 12 eight-month-old pups. Gene sequence analysis indicates that the virus is distinct from that of the previous, 1997, outbreak, although it falls within the same group as other canid isolates from northern South Africa. Since jackals (Canis mesomelas) are the principal host species of rabies in the area and domestic dogs do not occur in the Reserve, it is assumed that the jackal cycle was the source of the infection. Whereas the pups had not been vaccinated, the five adults, all of which survived, had each been vaccinated by the parenteral route at least twice and each had significant rabies serum neutralising antibodies. This indicates that multiple vaccination against rabies is effective in protecting wild dogs against challenge. Our findings extend previous observations that indicated failure of protection against rabies with single parenteral vaccinations in African wild dogs.