To eliminate the influence of anti-dog erythrocyte membrane antibodies in Babesia gibsoni-infected dogs on the immunological investigations of parasites, an attempt was made to purify B. gibsoni piroplasms from infected erythrocytes by N2 cavitation and centrifugation in a Percoll discontinuous density gradient. Although electron microscopic examination revealed that the concentrated piroplasms visible as a band at the interface between 30% and 40% Percoll solutions were contaminated with a small amount of erythrocyte membrane debris, little contamination was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblots. Hence, the isolated parasites may be usable for immunological examinations of parasites.