Vaccinia virus preparations agglutinate chicken erythrocytes and this hemagglutination is specifically inhibited by antivaccinial sera. Since the inhibition of helnagglutinanoit by antisera may be one of the most sensitive reactions as a method for detecting the specific antibody, it seems probable that this reaction may be applied to the diagnosis for variola.In the present study, we, first, investigated various factors having relations to the hemagglutination by vaccinia virus materials and its inhibition by specific antisera in order to look for the optimal conditions deciding the standard procedure, and then, searched the inhibiting antibodies in the sera of vaccinated persons or variola patients for the purpose of determining whether the hemagglutination inhibition is worthy of use for variola diagnosis.The results were as follows:I. Investigations of the factors relating to the hemagglutination and the inhibition1) The diluting medium was one of the most important factors affecting the regularity of the reaction. The findings showed that 0.1% normal chicken plasma in physiological saline might be one of the most suitable diluting media to expect a regular reaction constantly.2) Either testicular lapine preparations or suspensions of chorioallantoic membranes of developing chicken embryos infected with vaccinia virus were available as the hemagglutinin materials. The hemagglutinin titers of these preparations were about 5×107-5×109.Cow lymph could not agglutinate chicken red cells.When the virus preparations were stored in a refrigerator, their hemagglutinin titers remained essentially unchanged for a month, and when inactivated by treating with 0.05% formalin, 0.05% phenol, 0.01% merzonin (sodium ethylmercurithiosalicylate) or by ultraviolet irradiation, the titers were reduced only in a small quantity.3) The differences in susceptibility to agglutinating effects of vaccinia virus materials were observed amongst individual chicken red cells. The optimal quantity of red cells was 0.125% in the final concentration.4) The hemagglutination by vaccinia virus preparations was best shown at 37°C. The results could be read 1 1/2-2 hours after the incubation.5) The available procedure of hemagglutination was carried out as follows. Serial dilutions of serum and 8 agglutinating doses of virus preparation were mixed, incubated at 37°C for 30 to 60 minutes, and 0.25% suspension of red cells being subsequently added. Then, the mixtures were again incubated at 37°C for 1 1/2-2 hours before being read.