We compared 24 rabies samples collected in Estonia in 1989 to 1992, to identify the kinds of rabies strains circulating in this country. Eleven of the strains came from the islands of Saaremaa, Hiiumaa and Muhu, off the Baltic coast; 13 came from the mainland. The mainland strains, like those from the 1988 to 1989 epizootic in Finland, were antigenically different from the 11 island isolates. The island isolates reacted negatively with monoclonal antibody W-187.5 as does the SAD B19 rabies vaccine strain, currently spread as baits to wildlife in Finland and other parts of Europe. In order to unambiguously distinguish the island isolates from the SAD B19 vaccine, we developed a polymerase chain reaction (PCR) protocol for rabies, followed by a single restriction enzyme digestion. This method enabled the island isolates to be differentiated with ease from the vaccine strain SAD B19 at the level of the nucleoprotein-coding region. Additionally, this method had the ability to distinguish other polar field isolates examined, as well as the laboratory challenge virus strain CVS, from SAD B19 vaccine. Modifications of the above PCR method may be used for epidemiological investigations of new outbreaks or of outbreaks involving different species.