An African (Kenyan) strain of Babesia bigemina, Muguga (B2-1), was inoculated into a calf from a stabilate and blood from the calf was used to establish the parasite in vitro. The strain has been cultured continuously for 20 months, initially in bovine erythrocytes with 60% adult bovine serum, later, with 50%. Cultures were incubated at 37???C in RPMI 1640 medium with a gas mixture of 1% O2, 5% CO2, 94% N2. Adaptation in?vitro was demonstrated when serum from a calf which had recovered from infection with B2-1 bound to proteins of Mr 46?kDa, 49 kDa, 52 kDa, 61?kDa and 72?kDa on Western blots of B2-1 antigens from cattle blood but did not recognise the 49 kDa or 52 kDa antigens from in-vitro-derived parasites. These proteins were considered specific for B2-1, as they were not recognised by the same serum on profiles of a Mexican isolate of B. bigemina or an African isolate of B. bovis (Kwanyange). After 9 months of in vitro culture, a stabilate of the cultured parasite was injected into two splenectomised calves and one intact calf. The calves experienced a drop in packed cell volume and low parasitaemias but recovered spontaneously. Two of these animals, one splenectomised and one intact, were challenged with virulent B2-1 and experienced only mild babesiosis, in contrast to a previously uninfected calf also challenged with B2-1, which had to be euthanised after 5 days with severe babesiosis.