The primary structure of the alpha and beta subunits of phosphorylase kinase reveals that both proteins contain a carboxyl-terminal CA1A2X motif (where C is cysteine, A1 and A2 are aliphatic amino acids, and X is an uncharged amino acid), the recognition signal for a protein polyisoprenyltransferase. The product, a polyisoprenylated cysteine, can be detected by phenylthiocarbamoylamino acid analysis and by microsequencing following conversion to S-ethylcysteine. Mass spectrometry confirms a covalently linked farnesyl residue in both subunits. Tandem mass spectrometry localizes these modifications at the cysteine residues present in the carboxyl-terminal CAMQ and CLVS sequences of the alpha and beta subunits, respectively. Membrane association of phosphorylase kinase, probably mediated by these farnesyl residues, is discussed.