An effective axenic culture system for Trypanosoma brucei rhodesiense (ILRAD 1501) bloodstream forms is demonstrated. Bloodstream forms were continuously grown in 25 mM HEPES-buffered D-MEM supplemented with 10 microM bathocuproine sulfonate (BCS), 100 microM cysteine, and 20% heat-inactivated fetal bovine serum at 37 degrees C in vitro. At the initiation of the culture, T. b. rhodesiense bloodstream forms required the presence of 0.2 IU/ml insulin and 1 mM pyruvate, while bloodstream forms were grown in the culture medium without these supplements 4 days after initiation of the culture. Under this culture condition, T. b. rhodesiense bloodstream forms increased in number to 7 to 8 x 10(6) trypanosomes/ml, by day 4 after initiation of the culture. The trypanosomes cultured in this axenic system for 150 days were typically long and slender and retained their virulence for mice. This axenic culture system is extremely useful for in vitro cloning of T. b. rhodesiense bloodstream forms in vitro.