The reverse transcriptase polymerase chain reaction (RT-PCR) assay was used to detect Equine Arteritis Virus (EAV) in the semen of 88 horses and 2 donkeys, with neutralising antibodies against EAV, on the basis of the amplification of a 279 bp long fragment located in the viral polymerase gene. The RT-PCR assay revealed the virus at 4 TCID50/ml in cell culture and showed a greater sensitivity (54.4%) than cell culture isolation (33.3%). Moreover, the two samples of donkey semen were found positive. The cDNAs obtained from 14 samples of horse and 2 of donkey semen were sequenced. Comparing the sequence of reference strain Bucyrus, the analysed samples were 78-100% identical and showed a 84-97% nucleotide identity with Bucyrus isolate. The results demonstrate high levels of genomic heterogeneity among the extracted RNAs, but inside the fragment amplified a well-preserved region of 24 bp was found with only three mismatches in some samples, suggesting that this could be ideal as a probe for RT-PCR-ELISA. The RT-PCR-ELISA assay using the EAV 7 and 8 primer set, has proved to be sensitive, specific and above all directly applicable to semen. Additionally, the short time needed for the overall procedure makes this method suitable for diagnostic purposes.