The nucleotide analogue 5-p-fluorosulfonylbenzoyladenosine reacts with rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase kinase, causing a rapid loss of the AMP activation capacity and a slower inactivation of the catalytic activity. The rate constant for loss of AMP activation is eleven times higher (K1 = 0.107 min-1) than the rate constant for inactivation (K2 = 0.0094 min-1). Mg-ATP protects preferentially against inactivation, while Mg-AMP at a low concentration (7.5/0.05 mM) protects preferentially against loss of the AMP activation capacity. Oppositely, Mg-ADP at a low concentration (7.5/0.05 mM) hardly protects against loss of AMP activation capacity. We conclude that microsomal reductase kinase has distinct sites for activation and catalysis.