The protozoan parasite Babesia bovis, a causative agent of bovine babesiosis, has been continuously cultivated in a settled layer of bovine erythrocytes. Lowered oxygen tension within the layer of host erythrocytes results in a darkening of infected cultures and provides a rapid means of evaluating parasite growth. Deprivation of carbon dioxide causes the merozoites to accumulate in the medium rather than involving new erythrocytes. When separated from the culture, these extraerythrocytic parasites retain their infectivity. Parasites produced in vitro are morphologically identical to parasites from the blood of infected cattle and are susceptible to antibabesial drugs.