Groups of five dogs were immunized with vaccines containing soluble parasite antigens (SPA) derived from in vitro culture of Babesis canis parasites, either obtained commercially (Pirodog) or produced at laboratory scale. Both vaccines generated antibodies that reacted with parasitised erythrocytes (PE). Upon challenge infection with homologous parasites, protection was evident from less severe decreases of haematocrit values, and reduced morbidity. Vaccinated animals, however, were not protected against challenge infection with heterologous B. canis parasites. Recovery from challenge infection coincided with the production of antibodies against parasitized erythrocytes. The results suggest that SPA from B. canis carry strain-specific determinants that are crucial for inducing protection in dogs against challenge infection, and explain vaccination failures in the field.