For the isolation of Brucella ovis, modified Thayer-Martin medium with the addition of 0.01 micrograms/ml of trimethoprim and 100 IU of nystatin (TMM) was compared with Skirrow Agar (SK). Using viable counting technique, 11 strains were evaluated and the results were compared with those using Columbia Base Agar with bovine blood 7% (CBA). Ninety-four semen samples of 33 rams from a flock with infection antecedents were cultured on the same media. Growth of Brucella ovis strains was similar in all three media with the exception of one strain that did not grow on TMM. The results of semen cultures were the same for TMM and SK media and B. ovis was isolated from 27% of the samples. The results indicate that TMM and SK media are excellent for the isolation of B. ovis from semen of rams in field conditions.