The type I signal peptidase lepB genes from Rickettsia rickettsii and Rickettsia typhi, the etiologic agents of Rocky Mountain spotted fever and murine typhus, respectively, were cloned and characterized. Sequence analysis of the cloned lepB genes from R. rickettsii and R. typhi shows open reading frames of 801 and 795 nucleotides, respectively. Alignment analysis of the deduced amino acid sequences reveals the presence of highly conserved motifs that are important for the catalytic activity of bacterial type I signal peptidase. Reverse transcription-PCR and Northern blot analysis demonstrated that the lepB gene of R. rickettsii is cotranscribed in a polycistronic message with the putative nuoF (encoding NADH dehydrogenase I chain F), secF (encoding protein export membrane protein), and rnc (encoding RNase III) genes in a secF-nuoF-lepB-rnc cluster. The cloned lepB genes from R. rickettsii and R. typhi have been demonstrated to possess signal peptidase I activity in Escherichia coli preprotein processing in vivo by complementation assay.