The assay of Rickettsia tsutsugamushi infectivity by plaquing has been improved substantially by a number of changes which were based on our understanding of factors which enhance scrub typhus rickettsial infection of, and replication in, cultured cells. Greater numbers of plaques and/or larger plaques resulted from: 1) use of tissue culture medium instead of brain heart infusion broth as the rickettsial diluent; 2) plaquing in a contact-inhibited mouse embryo cell line rather than in growth-inhibited or uninhibited Vero cells; 3) infection and incubation of monolayers at 35°C instead of at lower temperatures; 4) frequent feeding of infected cultures with medium containing ample amounts of serum; and 5) inclusion of chicken serum in the overlay medium. Plaquing in 24-well tissue culture plates instead of in petri dishes or flasks greatly simplified the handling of large numbers of samples and was beneficial economically as well. Easily recognized rickettsial plaques were counted microscopically under ×40 magnification, and maximum counts were obtained 12–14 days after infection, depending on the rickettsial strain. Slightly longer incubation yielded macroscopically visible counts. In addition to enhancing plaque number and size, the changes in standard R. tsutsugamushi plaquing methods resulted in an easier, faster, and more reliable assay, with improved reproducibility of plaque formation, maintenance of infected cell monolayers, and avoidance of microbial contamination.