The infection cycle of Rickettsia rickettsii, studied in slide chamber cultures of chicken embryo and L-929 cells, was found to be complex and did not conform to a one-step growth cycle. Initial uptake kinetics resembled those established for Rickettsia prowazekii, but subsequent events showed very marked differences. Intracytoplasmic growth commenced exponentially without measurable lag. However, very soon after infection, intracytoplasmic rickettsiae began to escape from the host cell into the medium in large numbers, resulting in (i) failure of large numbers of rickettsiae to accumulate in the cytoplasm, (ii) sustained rapid division of the organisms in the cytoplasm, (iii) substantial accumulation of extracellular rickettsiae, and (iv) rapidly spreading infection in the culture, with most cells infected in 48 to 72 h. In the occasional cell, rickettsiae were found in the nucleus, where they multiplied to form compact masses. Thus, analysis of the growth characteristics of R. rickettsii must consider the entire culture as a unit in which the rickettsiae are distributed among three compartments in which they behave in different ways: (i) intranuclear, (ii) intracytoplasmic, and (iii) extracellular. The rickettsial traffic is bidirectional across the host cell plasma membrane and dominantly monodirectional across the nuclear membranes. The implications of this behavior with respect to location and range of receptors and substrates involved in membrane penetration are discussed. In older cultures, unique intracytoplasmic ring or doughnut colonies were common, indicating a change in the intracytoplasmic environment. The possible significance of the growth characteristics in cell culture to the characteristics of infection in humans and animals is discussed.