As the factors influencing the neutralization test of rickettsiae, the following ones have been given; mouse organs employed, unequal distribution of rickettsiae in organs and determination method of the result. The author studied the influences of these factors and other various conditions on the neutralization test. Mitani strain of Rickttsia tsutsugamushi isolated from the patients of Umayado disease in Kagawa prefecture was used as the test agent. The results were as follows: 1) In the present experiment, though some errors should be taken into consideration so far as animal experiment is concerned, it was concluded that reliable result would be obtained by careful selection of the outer conditions and inner factors. 2) As the outer conditions, the experimental temperature and the solution for dilution of rickettsial emulsion are given. The temperature under 10°C is suitable for treatment of R. tsutsugamushi, and glutamine-sucrose-phosphate buffer is superior as the dilution solution. 3) As the inner factors, those of rickettsia itself and those of immune sera were considered. In orser to make the distribution of rickettsiae equal, many livers and spleens were pooled and treated at the low temperature mentioned above. The selection of immune sera is important, and the neutralization test with rabbit immune sera showed the best result. 4) After some observation period of neutralization test, a few of the survived mice showed the syndrom of the disease. This result suggested that prolongation of observation period was necessary to exact determination of the result of neutralization test.