Rickettsia africae is a newly described species which causes African tick bite fever. Mediterranean spotted fever caused by R. conorii is endemic in the same regions of Africa as tick bite fever, and differentiation of the two syndromes by characterization of their etiological agents is important for epidemiological studies. R. africae and R. conorii are, however, difficult to distinguish, and therefore, our aim was to produce monoclonal antibodies to address this problem. Monoclonal antibodies were produced against R. africae by fusing splenocytes from BALB/C mice immunized with purified rickettsial organisms and SP2/0-Ag14 myeloma cells. A total of 355 hybridomas producing monoclonal antibodies to R. africae were identified by initial screening with six different antigens by microimmunofluorescence assay. A panel of 23 representative monoclonal antibodies were selected and subcloned. This panel was screened with a further 17 different spotted fever group (SFG) rickettsial reference antigens. Of these 23 monoclonal antibodies, 1 cross-reacted with only R. parkeri, whereas the others cross-reacted with more than two different antigens. Immunoblotting indicated that all the monoclonal antibodies were directed against the epitopes on two major high-molecular-mass heat-labile proteins, of which the molecular masses were 128 and 135 kDa, respectively. This monoclonal antibody panel was used successfully to identify R. africae in the blood culture of an infected patient, in infected cells within shell vials, and in infected ticks collected from Africa. Furthermore, the cross-reactivity of each SFG rickettsia with each of these 23 monoclonal antibodies was scored and was used to build a dendrogram of taxonomic relatedness between R. africae and the other SFG rickettsiae on the basis of Jaccard coefficients and unweighted pair group method with arithmetic mean analysis. The relatedness was generally consistent with that obtained by other methods of comparison.