Spotted fever group (SFG) rickettsiae are obligate intracellular prokaryotes that include tick-borne pathogens of vertebrates as well as nonpathogenic organisms living in symbiotic association with their tick hosts. We investigated the ability of SFG rickettsiae to move between and within host cells using tick cell culture and a SFG rickettsial isolate from a lone star tick (Amblyomma americanum) collected in Missouri. The isolate (MOAa), which is closely related to Rickettsia montana, grew in cell lines from the ticks Ixodes scapularis and Rhipicephalus appendiculatus. Transmission electron microscopy demonstrated that immediately following entry into tick cells, rickettsiae escaped from the host cell membrane, and intracellular rickettsiae came to lie in direct contact with host-cell cytoplasm. There was evidence of damage to the endoplasmic reticulum membrane which was broken down into vesicular structures. When rickettsiae exited host cells, host membrane stretched around them but was lost before re-entry. Use of a fluorescein-tagged monoclonal antibody to rickettsial outer membrane protein B and rhodamine-labeled phalloidin demonstrated association of actin tails with rickettsiae and suggested that SFG rickettsiae utilized host cytoskeletal components for movement. During early stages of infection, when cells harbored only one or a few organisms, “comet tails” of F-actin formed on one end of rickettsial cells, presumably pushing them ahead. Actin tails were not seen during later stages of infection when tick cells became completely filled with rickettsiae.