Actinobacillus seminis isolates were cultured from the semen (two isolates) and the left testis (one isolate) of two one-year-old rams in León, Spain. One animal showed lesions of chronic unilateral orchitis and epididymitis while the other appeared to suffer a subclinical infection and only a moderate number of pleomorphic rods and inflammatory cells were present in its semen. The isolates were biochemically similar to the A. seminis type strain NCTC 10851 and two other European A. seminis isolates, except that they produced acid from sorbitol; their identity was confirmed by arbitrarily primed polymerase chain reaction. The isolates were also tested against 30 antimicrobial agents, and only marbofloxacin was found active against all of them. As far as is known, this is the first report of A. seminis isolation from rams in southern Europe.