The maturation and quantification of Theileria lestoquardi (T. hirci) parasites in unfed and partially fed adult Hyalomma anatolicum anatolicum ticks was studied using (1) methyl green pyronin (MGP) staining of salivary glands, (2) in vitro infection of peripheral blood mononuclear cells (PBM) with parasites harvested from infected ticks and (3) a semi-quantitative polymerase chain reaction (PCR). With MGP staining the greatest infection rate was seen in unfed ticks. Feeding resulted in a gradual reduction in the number of infected acini with a concomitant increase in the maturity of the parasites. In vitro infection of sheep PBM with titrated ground-up tick supernate (GUTS) demonstrated that infectivity peaked between 2 and 4 days of tick feeding whereas GUTS prepared from unfed ticks was not infective. The polymerase chain reaction (PCR) was both sensitive and specific, detecting T. lestoquardi DNA in unfed and partially fed ticks, with a maximum sensitivity of 0·022 infected acinus/tick in 2-day fed ticks, though it gave no indication of the infectivity of the parasite.