Approximately 75 individuals from a population of 111 Ethiopian wolves (Canis simensis) died or disappeared from the Bale Mountains National Park (Ethiopia) between 1988 and 1992 during two significant population declines. Confirmation of rabies virus in two carcasses was based on the fluorescent antibody test (FAT) and the mouse inoculation test (MIT). In an Ethiopian wolf brain previously designated rabies negative by both FAT and MIT, rabies virus was identified by nested reverse transcription-polymerase chain reaction (RT-PCR) and confirmed by Southern blot hybridization. These methods were successfully used on a highly decomposed brain sample which had been stored in 20% dimethyl sulfoxide. This test system allows early and sensitive detection to be undertaken to more effectively prevent spread of disease and thus protect surviving animals.