The heat shock protein RrmJ (FtsJ), highly conserved from eubacteria to eukarya, is responsible for the 2'-O-ribose methylation of the universally conserved base U2552 in the A-loop of the 23 S rRNA. Absence of this methylation, which occurs late in the maturation process of the ribosome, appears to cause the destabilization and premature dissociation of the 50 S ribosomal subunit. To understand the mechanism of 2'-O-ribose methyltransfer reactions, we characterized the enzymatic parameters of RrmJ and conducted site-specific mutagenesis of RrmJ. A structure based sequence alignment with VP39, a structurally related 2'-O-methyltransferase from vaccinia virus, guided our mutagenesis studies. We analyzed the function of our RrmJ mutants in vivo and characterized the methyltransfer reaction of the purified proteins in vitro. The active site of RrmJ appears to be formed by a catalytic triad consisting of two lysine residues, Lys-38 and Lys-164, and the negatively charged residue Asp-124. Another highly conserved residue, Glu-199, that is present in the active site of RrmJ and VP39 appears to play only a minor role in the methyltransfer reaction in vivo. Based on these results, a reaction mechanism for the methyltransfer activity of RrmJ is proposed.