A limiting dilution microculture system was optimized to quantify the frequency of Theileria parva-specific cytotoxic T lymphocyte precursors (CTLp) in peripheral blood mononuclear cells (PBMC) from immune cattle. Optimal results were obtained with responder cell input levels ranging from 2 x 10(4)/well to 6.25 x 10(2)/well, along with 1-5 x 10(3)/well stimulator cells in standard supplemented RPMI 1640 medium containing 2.5-5% T cell growth factors. Thirty-six microtitre wells were established at each responder input level. Cultures were incubated for 7 days at 38-degrees-C, at the end of which time individual wells were screened for cytotoxic activity in a 4-h indium-111 oxine-release assay. Analysis of the cytotoxicity data, by a computer-programmed maximum likelihood estimation method indicated that they conformed to the Poisson model of single-hit kinetics. Estimates of frequencies ranged from 1:3600 to 1:5275 CTLp in PBMC of eight cattle between 1 and 24 months after immunization with T. parva. By contrast, no CTLp were detected in six naive animals analysed to a responder cell input of 10(5)/well. Split-well analysis of individual microwells showed that the CTL clones generated under limiting dilution conditions displayed exquisite specificity for parasitized cells, were genetically restricted and in some animals were parasite strain-specific.