(S)-3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA), the physiologic substrate of HMG-CoA reductase and of HMG-CoA lyase, is available commercially only as (R,S)-HMG-CoA, a mixture of diastereomers. To provide (S)-HMG-CoA for our continuing investigation of HMG-CoA reductase, we used homogeneous, overexpressed Pseudomonas mevalonii HMG-CoA reductase (EC 1.1.1.88) and an NAD(+)-regenerating system to convert (R)-mevalonate to (S)-HMG-CoA with an overall yield in excess of 50%. We also used P. mevalonii HMG-CoA lyase (EC 4.1.3.4) to prepare (R)-HMG-CoA from (R,S)-HMG-CoA. Each diastereomer was then isolated by ion-exchange chromatography. Large-scale preparations provide for economical production of (S)-HMG-CoA, particularly when recovered coenzyme A is recycled. (S)-HMG-CoA was evaluated as a substrate, and (R)-HMG-CoA as an inhibitor, for the P. mevalonii enzymes HMG-CoA reductase and HMG-CoA lyase, and for Syrian hamster HMG-CoA reductase (EC 1.1.1.34). For both HMG-CoA reductases, (R)-HMG-CoA inhibited competitively with respect to (S)-HMG-CoA. The ratio Ki/Km was 0.7 +/- 0.1 and 0.6 +/- 0.2 for the bacterial and hamster enzymes, respectively. By contrast, (R)-HMG-CoA did not inhibit P. mevalonii HMG-CoA lyase.