The known amino acid sequences at the two sites on phosphorylase kinase that are phosphorylated by cyclic AMP dependent protein kinase were extended. The sequences of 42 amino acids around the phosphorylation site on the α subunit and of 14 amino acids around the phosphorylation site on the β subunit were shown to be: α subunit Phe Arg Arg Leu Ser(P) Ile Ser Thr Glu Ser Glx Pro Asx Gly Gly His Ser Leu Gly Ala Asp Leu Met Ser Pro Ser Phe Leu Ser Pro Gly Thr Ser Val Phe(Ser,Pro,Gly)His Thr Ser Lys; β subunit, Ala Arg Thr Lys Arg Ser Gly Ser(P) Val/Ile Tyr Glu Pro Leu Lys. The sites on histone H2B which are phosphorylated by cyclic AMP dependent protein kinase in vitro were identified as serine 36 and serine 32. The amino acid sequence in this region is: Lys Lys Arg Lys Arg Ser(P) Arg Lys Glu Ser(P) Tyr Ser Val Tyr Val [Iwai, K., Ishikawa, K, & Hayashi, H. (1970) Nature (London) 226, 1056 - 1058]. Serine 36 was phosphorylated at 50% of the rate at which the β subunit of phosphorylase kinase was phosphorylated, and it was phosphorylated 6-7 fold more rapidly than was serine 32. The amino acid sequences when compared with those at the phosphorylation sites of other physiological substrates suggest that the presence of two adjacent basic amino acids on the N terminal side of the susceptible serine residue may be critical for specific substrate recognition in vivo.