Members of the genus Rickettsia range from nonpathogenic endosymbionts to virulent pathogens such as Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever. Many rickettsiae are considered nonpathogenic because they have been isolated from ticks but not vertebrate hosts. We assessed the ability of three presumed endosymbionts: Rickettsia amblyommatis, Rickettsia bellii, and Rickettsia montanensis, to infect a guinea pig animal model. These species were chosen because of their high prevalence in respective tick vectors or published reports suggestive of human or animal pathogenicity. Following intraperitoneal (IP) inoculation of cell culture suspensions of R. rickettsii, R. amblyommatis, R. bellii, or R. montanensis into guinea pigs, animals were monitored for signs of clinical illness for 13 days. Ear biopsies and blood samples were taken at 2- to 3-day intervals for detection of rickettsial DNA by PCR. Animals were necropsied and internal organ samples were also tested using PCR assays. Among the six guinea pigs inoculated with R. amblyommatis, fever, orchitis, and dermatitis were observed in one, one, and three animals respectively. In R. bellii-exposed animals, we noted fever in one of six animals, orchitis in one, and dermatitis in two. No PCR-positive tissues were present in either the R. amblyommatis- or R. bellii-exposed groups. In the R. montanensis-exposed group, two of six animals became febrile, two had orchitis, and three developed dermatitis in ears or footpads. R. montanensis DNA was detected in ear skin biopsies collected on multiple days from three animals. Also, a liver specimen from one animal and spleen specimens of two animals were PCR positive. The course and severity of disease in the three experimental groups were significantly milder than that of R. rickettsii. This study suggests that the three rickettsiae considered nonpathogenic can cause either subclinical or mild infections in guinea pigs when introduced via IP inoculation.