The crystal structure of YecO from Haemophilus influenzae (HI0319), a protein annotated in the sequence databases as hypothetical, and that has not been assigned a function, has been determined at 2.2-Å resolution. The structure reveals a fold typical of S-adenosyl-L-methionine-dependent (AdoMet) methyltransferase enzymes. Moreover, a processed cofactor, S-adenosyl-L-homocysteine (AdoHcy), is bound to the enzyme, further confirming the biochemical function of HI0319 and its sequence family members. An active site arginine, shielded from bulk solvent, interacts with an anion, possibly a chloride ion, which in turn interacts with the sulfur atom of AdoHcy. The AdoHcy and nearby protein residues delineate a small solvent-excluded substrate binding cavity of 162 Å3 in volume. The environment surrounding the cavity indicates that the substrate molecule contains a hydrophobic moiety and an anionic group. Many of the residues that define the cavity are invariant in the HI0319 sequence family but are not conserved in other methyltransferases. Therefore, the substrate specificity of YecO enzymes is unique and differs from the substrate specificity of all other methyltransferases sequenced to date. Examination of the Enzyme Commission list of methyltransferases prompted a manual inspection of 10 possible substrates using computer graphics and suggested that the ortho-substituted benzoic acids fit best in the active site. Proteins 2001;45:397–407. © 2001 Wiley-Liss, Inc.