There are two classes of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase: the class I enzymes of eukaryotes and some archaea, and the class II enzymes of certain eubacteria. The activity of the class I Syrian hamster HMG-CoA reductase is regulated by phosphorylation-dephosphorylation of Ser871. Phosphorylation apparently prevents the active site histidine, His865, from protonating the inhibitory coenzyme A thioanion prior to its release from the enzyme. Structural evidence for this hypothesis is, however, lacking. The HMG-CoA reductase of the thermophilic archaeon Sulfolobus solfataricus, whose stability recommends it for physical studies, lacks both a phosphoacceptor serine and a protein kinase recognition motif. Consequently, its activity is not regulated by phosphorylation. We therefore employed site-directed mutagenesis to engineer an appropriately located phosphoacceptor serine and cAMP-dependent protein kinase recognition motif. Substitution of serine for Ala406, the apparent cognate of hamster Ser871, and replacement of Leu403 and Gly404 by arginine created S. solfataricus mutant enzyme L403R/G404R/A406S. The general properties of enzyme L403R/G404R/A406S (K(m) values, V(max), optimal pH and temperature) were essentially those of the wild-type enzyme. Exposure of enzyme L403R/G404R/A406S to [gamma-(32)P]ATP and cAMP-dependent protein kinase was accompanied by incorporation of (32)P(i) and by a parallel decrease in catalytic activity. Subsequent treatment with a protein phosphatase released enzyme-bound (32)P(i) and restored activity to pretreatment levels. The regulatory properties of enzyme L403R/G404R/A406S thus match those of the hamster enzyme. Solution of the three-dimensional structures of the phospho and dephospho forms of this mutant enzyme thus should reveal structural features critical for regulation of the activity of a class I HMG-CoA reductase.