The protozoan parasite Babesia equi, a causative agent of equine piroplasmosis, was continuously cultivated in horse erythrocytes. The parasites were isolated from a carrier horse at a time when no parasite was detected in a thin blood smear. The culture medium consisted of modified medium 199 supplemented with 40% non-heat-inactivated horse serum in a humidified atmosphere containing 5% CO2, 2% O2, and 93% N2 at 37 degrees C. Parasites were detected after 2 days in culture. When the percentage of parasitized erythrocytes (PPE) reached 1%, the cultures were transferred into a humidified atmosphere of 5% CO2 in air. After 7 days the cultures were split at a ratio of 1:2, and after another 5 days they were split at a ratio of 1:4. From them on, cultures were split at a ratio of 1:4 routinely at 2-day intervals. The PPE ranged between 10% and 25%. Supplementation with hypoxanthine was essential for the initiation and propagation of cultures. In established cultures, hypoxanthine could be replaced by equimolar concentrations of adenosine or guanosine. Parasites from cultures could be cryopreserved and resuscitated. Cultures were maintained for more than 300 days.