In vitro cultivation of Babesia rodhaini (BR) and Babesia microti (BM) was attempted. When RPMI1640 was supplemented with 30 or 40% of non-treated fetal bovine serum (FBS), the gas mixture of 3% CO2-8% O2 best supported the growth of both parasites. Under this optimized condition, the percent parasitized erythrocytes peaked to approximately 4- and 2-times initial values for Br and BM, respectively. The cultivated parasites retained the infectivity to the host mice. BM showed the characteristic feature of division during cultivation. However, the lots of FBS will have to be taken into consideration, since the FBS lots were shown to give large varieties to the results. Selection of the appropriate FBS lot may yield the better growth of these protozoa.