Babesia ovis, the primary causative agent of ovine babesiosis, is a tick-borne hemoprotozoan parasite and causes serious losses in small ruminant livestock in tropical and subtropical regions. The current control of ovine babesiosis has been base on chemotherapy and limited tick control measures. Attenuated parasites which are valuable sources of live vaccines have been used to prevent babesiosis in cattle. There is a necessity to the studies about prevention of the disease by means of immunoprophylaxis in sheep. For this purpose, this study was planned to obtain the attenuated derivatives of a virulent B. ovis strain by successive blood passages in splenectomized lambs. A total of seventeen, six-month-old lambs, 13 of which splenectomized, 4 of which spleen intact lambs, known to be negative for the presence of hemoparasites and anti- B ovis antibodies were used. The splenectomized lambs were used for passaging the virulent B. ovis field strain. Four intact lambs divided into two groups were used to evaluate the pathogenicity of the virulent strain and twelfth passage strain of B. ovis. Blood collected from a clinically evident case of babesiosis in the field was inoculated intravenously into the first splenectomized lamb, and then the acute clinical disease was occurred. The same process was successively applied to the rest of 12 splenectomized lambs to have the attenuated B. ovis strain. The clinical, microscopic and hematological changes after inoculation were recorded in each passage of the lambs. All of the passage lambs were exhibited the severe symptoms of the disease from the 2nd day of inoculation. The same treatment protocol including vitamin B12 and the anti-babesial drug imidocarb dipropionate (IMDP) was applied to all splenectomized lambs on the fourth or fifth day of the inoculation. Although the symptoms of the disease were less severe than those of splenectomized lambs; two intact lambs infected with the virulent strain and two intact lambs infected with the twelfth passage blood were also showed the clinical and hematological findings of the disease on the 2nd and 3rd day after inoculation. Infection-related symptoms in all intact lambs disappeared spontaneously from the 6th day of inoculation without the application of any drug. The results of the study showed that there was no change on the virulence of B. ovis through the 12 successive blood passages. Therefore, the further passages or alternative attenuation methods may be necessary to attenuate the virulent strain of B. ovis. © 2013 Elsevier B.V.