Rabbits were immunized with free-living amoebas by intravenous injections. The amoebas were Acanthamoeba culbertsoni and Naegleria fowleri and obtained by axenic cultivation in CGVS medium. Each rabbit received 10(6) of Acanthamoeba culbertsoni and 10(5) of Naegleria fowleri trophozoites respectively every other day in three doses and finally one booster dose at 1 week later. Antiserum was collected from thc following day of the booster injection up to 2 months period, and stored at -30 degrees C until use. The immobilization test was performed. One drop of amoeba suspension was mixed with the test serum on slide and observed the mobile state under microscope. 1. Maximal immobilizing phenomenon observed in 30 minutes and, then gradually recovered to normal state. 2. Inactivation of antiserum at 56 degrees C for 30 minutes did not affect the immobilization phenomenon. 3. The immobilization rates decreased by the serial dilution of antiserum. At dilution more than 1:8, the immobilization was almost the same as in the normal serum. 4. The immobilizing antibody in anti-Acanthamoeba culbertsoni rabbit serum showed highest titre in 3rd day after booster immunization and from first to 6th week in anti-Naegleria fowleri rabbit serum. 5. Cross matching of Acanthamoeba culbertsoni and Naegleria fowleri showed antigenic difference of the two species. It is suggested that the immobilization reaction may be of value as a supplementary test in the diagnosis of primary amoebic meningoencephalitis.