Larval antigen of Hyalonmma anatolicum anatolicum, the vector of Theileria annulata, was purified by two-step affinity chromatography using anti-tick gut-specific rabbit IgG and IgG from immunized cattle. The purified antigen showed the presence of a single polypeptide of 37 kDa (GHLAgP) on SDS-PAGE. Two groups (I and II) of naive crossbred calves (Bos taurus x B. indicus) were immunized with I mg of GHLAgP in three divided doses. Immunized calves of group I were also infected with a sublethal dose of T annulata along with a group of non-immunized calves (group III). Animals in groups I, II, III as well a control group (group IV) were challenged with live nymphs of H. a. anatolicum on the 10th day of immunization. There was a significant reduction in the number of emerging adults of 56.9% +/- 1.67%, in calves of group I (p < 0.01) and 63.09% +/- 1.26% in calves of group II (p < 0.001) compared to the controls. The calves of groups I and II showed antibody responses to tick antigen up to day 70 post immunization. Infection with T. annulata was determined in the salivary glands of adult ticks that developed from the nymphs used for challenge infection. In ticks taken from group I calves, there was a 75.0% +/- 0.00% infection compared with only 85.0 +/- 2.88% infection in ticks taken from calves of group III. Using PCR, a lower infection (83.33% +/- 3.33%) was detected in ticks that developed from calves of group I compared with calves from group III (90.00% +/- 2.88%). The ground-up tick supernatants (GUTS) of the ticks taken from calves of group III yielded higher infection rate and exhibited higher infectivity titre in in vitro infection assay of bovine mononuclear cells than the GUTS of the ticks taken from calves of group I. The results suggest a partial reduction in growth rate of T. annulata in ticks feeding on calves immunized with GHLAgP.