A RT-PCR assay based on specific amplification of RNA sequences from each of the etiological agents of three important vesicular diseases that affect swine, foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and vesicular stomatitis virus (VSV), was developed. Genotype-specific primers that amplified DNA fragments of differential size from SVDV 3D gene or VSV L gene were selected with the aid of a computer program. Experimental testing of the primers predicted as SVDV-specific identified a primer pair, SA2/SS4, that rendered a specific product from SVDV RNAs, but did not amplify RNA from either FMDV or coxsackie B5 virus (CV-B5), a highly related picornavirus. Primers SA2/SS4 were used in combination with primers 3D2/3D1, which amplify a product of different size on FMDV 3D gene (Rodriguez et al., 1992). This combined RT-PCR reaction allowed a sensitive and specific differential detection of FMDV and SVDV RNAs in a single tube, by means of the analysis of the amplified products in agarose gels. The results obtained were similar when RNA extracted from viral stocks or plastic wells coated with either viral supernatants or extracts from lesions of infected animals, were used as starting material in the reactions. Using a similar approach, VSV serotype-specific primers IA/IS and NA/NS were selected for the specific amplification of VSV-Indiana and VSV-New Jersey RNAs, respectively. The combined use of SVDV, FMDV and VSV specific primers in a single reaction resulted in a genotype-specific amplification of each of the viral RNAs. Thus, differential diagnosis of FMDV from SVDV and/or VSV can be carried out in a single RT-PCR reaction, using a rapid and simplified methodology.