The inhibition of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase from yeast was compared for both F-244 (1) and (RS)-β -butyrolactone (5). F-244 exhibits irreversible inhibition with an IC50 of 8 nM, similar to that reported for the rat liver enzyme, while the binding constant (1/K1) and inactivation rate constant (kinact) similar to values reported for the human cytoplasmic enzyme. (RS)-β-Butyrolactone (5) also irreversibly inhibits the enzyme, but with much lower efficiency (IC50 2 mM). The values for K1 (9 mM) and kinact (0.0078 s−1) for 5 were determined. The results show that kinact for 5 and 1 differ by a factor of only 2.5, while K1 for 5 is higher by a factor of 1.8 × 105. Hence, the β-lactone ring is shown to be the sole essential structural feature in 1 for irreversible inactivation of HMG-CoA synthase; however, the remaining functionality enhances the binding of 1 to the enzyme relative to 5. Key words: HMG-CoA synthase, F-244, butyrolactone, irreversible, inhibitor.