Both (E)- and (Z)-phosphoenolbutyrates (FEB) were substrates for UDP-GlcNAc enolpyruvyl transferase, MurZ, the first enzyme of bacterial cell wall biosynthesis, and were converted to a mixture of (E)- and (Z)-enolbutyryl-UDP-GlcNAc (EB-UDP-GlcNAc). The catalytic rates for both isomers of FEB were about 0.2% that of the natural substrate, phosphoenolpyruvate (PEP). The initial kinetic ratio of (E)- to (Z)-enol ether products, 13:1, was similar for both (E)- and (Z)-PEB. The enzyme was thus stereoselective for the formation of the (E)-enol ether product but not stereospecific. On the other hand, the equilibrium ratio of (E)- to (Z)-enol ether products was 1:35; the thermodynamic product, (Z)-EB-UDP-GlcNAc, was different from the kinetic product, (E)-EB-UDP-GlcNAc. The overall equilibrium constant between (Z)-PEB and UDP-GlcNAc and (Z)-enol ether product and phosphate was approximately 65. (E)-PEB was also converted under initial velocity conditions to (Z)-PEB in addition to enol ether products, reflecting partitioning from an intermediate. In contrast, (Z)-PEB was not converted to a detectable amount of(E)-PEB. In D2O, (E)-[H-1]PEB was transformed to (Z)-[H-2]enol ether product and (E)-[H-1]enol ether product. Therefore, we concluded that the enzyme catalyzes either a syn-addition of UDP-GlcNAc to FEB and an anti-elimination of phosphate from an intermediate or an anti-addition and a syn-elimination. This is similar to the paired stereochemical alternatives in the reaction catalyzed by 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase).