The use of cyclophosphamide-treated mice for purpose of cloning the African trypanosomes was assessed. C3HeB/FeJ mice were injected with 200 mg/kg cyclophosphamide (CY) 24-72 hours prior to infection with a single trypanosome isolated from Trypanosoma rhodesiense infected blood samples. All CY-treated mice exhibited depressed parasite-exposure, which was a period of time sufficient to grow trypanosomes from a single organism to a fulminating parasitemia. Cloning efficiency was routinely 45% in these animals. Thus, our study demonstrates that CT-treated mice are a convenient and efficient vehicle for cloning African trypanosomes. Techniques which facilitate the selection of single trypanosomes from infected blood are also described in this report.