Rickettsiae are classified in the order Rickettsiales and have been included in the alpha subclass of the class Proteobacteria on the basis of 16S rRNA gene sequence comparison. To estimate the evolutionary forces that have shaped the members of the spotted fever group (SFG) rickettsiae, the ompA gene (apart from the tandem repeat units), encoding an antigenic high-molecular-mass membrane protein specific for the group, was amplified and sequenced from 21 isolates. The phylogenetic relationship between SFG rickettsiae were inferred from the comparison of both the gene and derived protein sequences, using the parsimony, neighbor-joining and maximum-likelihood methods. Three strongly supported phylogenetic sub-groups were distinguished: first, the Rickettsia conorii complex (R. conorii Malish, R. conorii M1, R. conorii Moroccan, R. conorii Indian tick typhus, Astrakhan fever rickettsia and Israeli tick typhus rickettsia); second, a cluster including Rickettsia africae, strain S, Rickettsia parkeri, Rickettsia sibirica and 'Rickettsia mongolotimonae'; and, third, a cluster including Rickettsia aeschlimannii, Rickettsia rhipicephali, Rickettsia massiliae, Bar 29 and Rickettsia montanensis. Rickettsia rickettsii, Rickettsia japonica, Rickettsia slovaca and Thai tick typhus rickettsia did not cluster with any other Rickettsia species. To test whether positive selection was responsible for sequences diversity, rates of synonymous and nonsynonymous nucleotide substitutions were compared for Rickettsia ompA alleles and indicated that this gene is undergoing neutral evolution.