A reverse transcription–polymerase chain reaction (RT–PCR), that uses primers specifically designed to amplify a portion of the N gene of vampire bat strains of rabies that circulate in Mexico, but also recognizing most of the rabies variants circulating in endemic areas, was established. This standardized PCR assay was able to detect viral RNA in tenfold serial dilutions up to a 107 dilution using stock virus at an original titre of 107·5 LD50. The assay was highly specific for rabies virus. Forty different rabies isolates recovered from different species and geographical regions in the country were diagnosed as positive and negative by the fluorescent antibody test (FAT). These same samples were re-examined by both PCR and the mouse inoculation test (MIT). Compared with MIT the PCR exhibited an epidemiological sensitivity of 86% and a specificity of 91% while its positive predictive value was 96%.