Experiments were undertaken to develop a serum-free medium for the in vitro cultivation of Babesia caballi, a tick-borne hemoprotozoan parasite, one of the causative agents of equine piroplasmosis. A modified HL-1 medium supplemented with horse serum, L-glutamine, antibiotics, and hypoxanthine was used. B. caballi organisms were continuously cultivated at 37 degrees C in microaerophilous stationary-phase culture in a humidified atmosphere containing 5% CO2 in air before exposure to serum-free culture conditions. For serum-free propagation, lipid-rich bovine serum albumin (LR-BSA), alone or with chemically defined lipids (CDL), were added instead of serum. Media containing LR-BSA alone or LR-BSA and CDL in various amounts supported the in vitro propagation of B. caballi. Growth was maintained for more than 6 months. The growth rates obtained in serum-free media were similar to those previously obtained in traditional serum-containing medium.