A plaque reduction assay for scrub typhus rickettsiae was used to study the antigenic relationships among the Gilliam, Karp, and Kato strains of Rickettsia tsutsugamushi. Antisera were prepared in rabbits, using single injections of cloned rickettsiae propagated in L-929 cells. Organisms grown in the yolk sac of embryonated chicken eggs were reacted with 4-fold dilutions of heat-inactivated immune sera, and plaqued in irradiated L-929 cells. Rabbit antiserum against each cloned strain of rickettsiae was effective in reducing the number of plaques of antigenically homologous organisms. Plaque reduction of heterologous antigenic strains of R. tsutsugamushi differed, depending on the antiserum employed. Gilliam and Karp antisera reacted weakly against the heterologous eliciting organism, but both antisera were effective in the plaque reduction of Kato. Kato antiserum showed broad reactivity, and reduced the number of plaques formed by either Gilliam or Karp organisms. Immune sera from rabbits infected with the Gilliam strain were fractionated on sucrose gradients and both IgM and IgG were capable of causing plaque reduction of cloned Gilliam rickettsiae. The plaque reduction assay was useful in comparing the antigenic characteristics of strains of R. tsutsugamushi, and may provide data which will contribute to an antigenic classification of scrub typhus rickettsiae.