SummaryEighteen strains of Actinobacillus seminis isolated from ovine semen samples and epididymitis were characterized according to their cultural and biochemical behaviour. The semen samples were collected from young, unmated and mature breeding rams suffering from epididymitis or subclinical genital infection.All A. seminis strains were non‐motile, non spore‐forming, uncapsulated, Gram‐negative pleomorphic rods, approximately 1–8 μm × 0.8‐1 μm. On bovine blood agar they formed low convex, greyish‐white, non‐adherent and non‐haemolytic colonies, which grew under aerobic conditions, but more luxuriantly when cultures were incubated in an atmosphere containing 10% CO2 at 37°C.All strains were catalase, oxidase, nitrate and ornithine decarboxylase positive, but indole, urease, phosphatase and beta‐galactosidase (ONPG) negative. They were fermentative in the O/F test and usually produced acid within 1 to 3 days from glucose, arabinose, fructose, maltose and xylose, but not from lactose, mannose and sucrose.In the agar gel diffusion test, antigenic differences were demonstrated among the isolates. On the basis of the cultural, morphological and biochemical characteristics, A. seminis can be differentiated from other Gram‐negative bacteria (e.g. Brucella ovis, Histophilus ovis etc.) involved in genital infections in rams.