A 3-step procedure for cloning Theileria parva parasites was developed. The first step involved the in vitro infection of a fixed number of bovine lymphocytes with titrated sporozoites. The cell lines obtained from infections initiated using sporozoite/lymphocyte ratios below 1:100 were then selected for cloning as these contained schizont-infected cells, each of which was derived from infection with a single sporozoite. In the second step, these cell lines were cloned by limiting dilution. As sporozoites infect lymphocytes and transform to induce clonal multiplication, this step produced infected cell lines containing both cloned parasites and cloned lymphocytes. In the third step, the cloned cell lines were used to infect cattle and isolation of the parasite in ticks was made during piroplasm parasitaemia. Finally, sporozoites were harvested from infected ticks and used for further characterization. Sporozoites derived from cloned cell lines of T. parva Muguga, Marikebuni, Boleni, Uganda and buffalo-derived 7014 were characterized using monoclonal antibody profiles, DNA restriction fragment length polymorphism detected using repetitive and telomeric probes, in vivo infectivity and, in one case, cross-immunity studies. Additionally, several distinct schizont-infected lymphocyte clones were isolated from the Muguga, Mariakani and buffalo-derived 7014 stocks. The combined results of the characterization revealed that the cloning procedure selected clones of T. parva from the parental stocks which were known to contain a mixture of genetically different parasite populations. The cloning method and the clones generated will be of value in studies of the biology of the parasite and in elucidating the strain specificity of immune responses in cattle.