Babesia caballi merozoites were prepared by combining two improved methods of cultivation and purification of merozoites using Percoll-gradiation, and the protein compositions of merozoites were analyzed by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. The relative molecular masses of the major proteins and protein masses separated by electrophoresis were >94, 80-70, 50-45, 34-30, 30-28 and 18 kDa. By Western blotting, twelve proteins or protein groups were recognized by pooled sera from two horses experimentally infected with B. caballi. Among twelve proteins, five new proteins (54, 30-26, 24, and two 18 kDa) were identified, and the 48 kDa protein was revealed to consist of 2 components in the B. caballi merozoite. One protein (54 kDa) of B. caballi was also recognized by the pooled sera from two horses experimentally infected with B. equi.