This study describes polymorphism in Theileria annulata, an intracellular protozoan parasite of bovine leucocytes and red blood cells. Fifty-three different stocks of T. annulata, isolated from 17 sites (districts) in Tunisia, have been characterized by anti-parasite monoclonal antibody (MAb) reactivity, glucose phosphate isomerase (GPI) isoenzyme electrophoresis, and Southern blotting with two genomic DNA probes. There appears to be considerable diversity amongst T. annulata stocks from Tunisia, no two isolates being identical, even those from animals on the same farm. Two distinct antigenic populations were detected by MAb 7E7. They were defined by negative and positive cells in the indirect fluorescent antibody test. The percentage of positive cells in different isolates ranged between 0 and 100%. The population variation seen by GPI analysis and DNA probes was greater; 7 different GPI phenotypes were identified amongst the stocks studied, while DNA probes T. annulata Tunis (TaT) 17 and 21 detected up to 5 different variants. The majority of isolates were shown to contain more than one parasite population, the number of variants per isolate ranging from 1 to 4. No correlation between particular parasite phenotypes or genotypes and their geographical site of isolation was observed. Selection of parasite populations in vivo and in vitro is also discussed.