UDP-GlcNAc acyltransferase (LpxA), the first enzyme of lipid A biosynthesis, catalyzes the transfer of an acyl chain activated on acyl carrier protein (ACP) to UDP-GlcNAc. LpxAs are very selective for the lengths of their acyl donor substrates. Escherichia coli LpxA prefers R-3-hydroxymyristoyl-ACP to R-3-hydroxydecanoyl-ACP by a factor of approximately 1000, whereas Pseudomonas aeruginosa LpxA prefers the opposite. E. coli G173M LpxA and the reciprocal P. aeruginosa M169G LpxA show reversed substrate selectivity in vitro and in vivo, demonstrating the existence of precise hydrocarbon rulers in LpxAs.