We have previously shown that incubation of isolated hepatocytes with fructose leads to elevation of AMP and activation of the AMP-activated protein kinase. We now show that this treatment causes marked inactivation of HMG-CoA reductase. Using immunoprecipitation from the microsomal fraction of 3 2 P-labelled cells, we also show that this treatment leads to a 2.6-fold increase in the phosphorylation of the 100 kDa subunit of HMG-CoA reductase. Successive digestion of this 3 2 P-labelled subunit with cyanogen bromide and endoproteinase Lys-C confirmed that Ser-871 the site phosphorylated in cell-free assays by the AMP-activated protein kinase, was the only site phosphorylated under these conditions.