The complete amino acid sequence of the regulatory subunit of type II cAMP-dependent protein kinase from bovine cardiac muscle is presented. Primary fragments for the sequence determination were obtained by limited proteolysis with various proteases or by cleavage with cyanogen bromide. The sequence of the 400 amino acid residues has two homologous regions, strongly suggesting tandem gene duplication. The predicted secondary structure suggests the presence of 42% alpha-helix, 23% beta-strand, and 23 beta-turns. The molecular weight of the subunit, as derived from the sequence, is 45,084 including a phosphate group at residue 95. This is significantly less than earlier estimates based on NaDodSO4 gel electrophoresis and sedimentation experiments. The structure is discussed in terms of putative sites of interaction with cAMP and with the catalytic subunit.