Rickettsia rickettsii is the tick-borne etiologic agent of Rocky Mountain spotted fever. R. rickettsii is the prototypic spotted fever group rickettsia. Several other species, R. conorii, R. siberica, R. japonica, R. akari, and others cause diseases of lesser severity. Still other species in the spotted fever group, R. montana, R. peacockii, R. belli, and R. rhipicephali, are considered avirulent as they have never been associated with human disease nor do they cause overt disease in standard laboratory animals. The typhus group of rickettsia, typified by R. prowazeki, the agent of epidemic typhus, include some of the historically most devastating disease agents known to mankind. The typhus group also includes species of lesser or no virulence potential to humans. R. prowazeki and R. rickettsii are classified as Biodefense Catagory B and C agents, respectively. Rickettsia rickettsii is a member of the spotted fever group rickettsiae and the etiologic agent of Rocky Mountain spotted fever (RMSF). R. rickettsii is a small obligate intracellular Gram-negative organism maintained in its tick host through transovarial transmission. Infection with R. rickettsii occurs through the bite of an infected tick 33. Once the organism gains access to the host it is able to replicate within the host vascular endothelial cells and spread from cell to cell by polymerizing host cell actin. Damage to vascular endothelial cells by R. rickettsii leads to increased vascular permeability and leakage of fluid into the interstices causing the characteristic rash observed in RMSF. Infection with R. rickettsii results in a severe and potentially life threatening disease if not diagnosed and treated properly. While much is known about the progression of disease, the molecular mechanisms involved in the pathogenesis of RMSF are poorly understood. Strains of Rickettsia rickettsii vary dramatically in their virulence in animal model systems and severity of human disease. The obligate intracellular lifestyle of rickettsiae and the lack of tractable genetic systems make it difficult to identify genes involved in virulence. With the completed sequences of multiple rickettsial species, it has become possible to investigate differences between virulent and avirulent strains of rickettsiae through comparative genomics. In efforts to generate isogenic mutant pairs to definitively identify rickettsial virulence determinants, we used the mariner-based transposon mutagenesis system to generate a collection of R. rickettsii mutants. Direct sequencing of purified genomic DNA from each of the clones using primers from within the transposon delineated the precise insertion sites. Transposition sites were randomly distributed throughout the rickettsial genome. Random transposon mutagenesis is being applied to generate a library of mutants for analysis. In addition, plasmid transformation to introduce and complement genes is being developed. Fusions of various reporters to rickettsial genes of interest is ongoing. In addition, methods to directly knock out specific rickettsial genes are being explored.