Rickettsia prowazekii, the etiologic agent of epidemic typhus, is a potential biological threat agent. Lysine methylation of OmpB (outer membrane protein) of Rickettsia correlates with the bacterial virulence. However, no lysine methyltransferase from Rickettsia has been characterized. Bioinformatic analysis of the Rickettsia prowazekii genome revealed six potential lysine methyltransferases. Codon‐optimized cDNAs of the six potential methyltransferases were synthesized and subcloned into pET28a. The six proteins were then expressed in E. coli BL21(DE3) and purified by affinity chromatography on Ni‐NTA agarose. The methyltransferase activity was assayed by the methyl incorporation of radioactively labeled S‐adenosyl‐methionine. Two of the six putative methyltransferases showed time‐ and enzyme‐concentration‐dependent methylation of the recombinant AN and K fragments of OmpB from R. typhi. The stoichiometry of the methyl incorporation suggests methylation at multiple sites of both fragments. Western blot analysis using trimethyl‐lysine specific antibodies confirmed the trimethylation of lysines in recombinant fragments of OmpB by the methyltransferases. Enzymatic methylation of OmpB delineates the functions of these proteins, and may provide tools for the development of diagnostic reagents