The biosynthesis of the pimelate moiety of biotin in Escherichia coli requires two specialized proteins, BioC and BioH. However, the enzymes that have BioC- or BioH-like activities show remarkable sequence diversity among biotin-producing bacteria. Here, we report that the intracellular rickettsial pathogen Ehrlichia chaffeensis encodes two novel proteins, BioT and BioU, which functionally replace the E. coli BioC and BioH proteins, respectively. The desthiobiotin assays demonstrated that these two proteins make pimeloyl-acyl carrier protein (ACP) from the substrate malonyl-ACP with the aid of the FAS II pathway, through the expected pimeloyl-ACP methyl ester intermediate. BioT and BioU homologues seem restricted to the species of Ehrlichia and its close relative, Anaplasma. Taken together, the synthesis of the biotin precursor in E. chaffeensis appears to be catalyzed by two novel BioC- and BioH-like proteins.