Two in a group of five naturally seropositive donkey stallions were found to shed equine arteritis virus (EAV) in their semen as demonstrated by virus isolation. Direct intramuscular inoculation of sonicated semen from one virus-shedding stallion (S3) caused clinical disease in two donkeys from which virus was recovered and in which seroconversion was detected. Sexual transmission was confirmed in two mares mated to S3 when after a febrile response during which EAV was isolated from huffy coats and nasal and ocular exudates, both mares were found to have seroconverted. In-contact transmission in a susceptible stallion was demonstrated after its exposure to a sexually infected mare. The 3' end of the asinine virus was amplified directly from donkey semen with EAV-specific primers, and its nucleotide sequence was found to be homologous to that of the prototype Bucyrus virus isolated from horses. These results indicate that EAV and its disease transmission are analogous in donkeys and horses.