SUMMARY Babesia bovis was isolated from an experimentally infected calf (No. 1) and was maintained in vitro for 32 days by subculturing 14 times, using a total dilution of 192,000. A splenectomized calf was inoculated with subculture Babesia (isolate B). The agent (isolate C) was isolated and maintained in vitro for 17 days prior to inoculation of a third splenectomized calf. Babesia organisms (isolate D) were isolated and retained in vitro for 270 days. After 85 days in vitro, organisms from this third isolation (isolate D) were injected into a fourth splenectomized calf to test for infectivity. Responses of the three calves to cultural inoculation were similar to those responses that occurred from inoculation of infected bovine blood (calf 1). There was no observed change in the microscopic appearance of the Babesia in vitro with time.