Recovery of infectious Equine arteritis virus (EAV) from the semen of persistently infected Swedish stallions was attempted by classical cell culture isolation and by transfection of extracted total RNA. Whereas virus from semen samples stored for several months at -20 degrees C or from extended semen could only be recovered by transfection of extracted RNA, isolation in cell culture was achieved readily with fresh, unextended semen stored at -70 degrees C or directly used after sampling. In parallel, the viruses were examined in the variable region of the large glycoprotein GP5 by nested RT-PCR and direct nucleotide sequencing. The resulting sequences were placed into a large phylogenetic tree from this region, demonstrating that Swedish strains belonged to very diverse phylogenetic groups. This represents the first report of recovery of infectious EAV from archived semen samples by RNA transfection.