A system has been described in which the penetration of Rickettsia tsutsugamushi (Karp strain) into tissue culture cells can be quantitated, and the factors affecting this process studied. The results indicated that rickettsial penetration in vitro depended largely on the viability of the organisms. Certain components of the fluid environment such as the divalent cations and protein were found to be of importance. The temperature dependence of the penetration process was found to vary with the nature of the suspending medium. A number of compounds related to L-glutamic acid enhanced penetration, whereas metabolic inhibitors depressed this process. Aureomycin at concentrations between 50 and 250 microg./ml. inhibited the penetration of rickettsiae while chloramphenicol at similar concentrations was ineffective. The results are discussed in terms of the biological and biochemical properties of this group of agents.