Treatment of cGMP-dependent protein kinase with low concentrations of trypsin generates an enzyme fragment of 65 kDa which is fully active in the absence of cGMP. The fragment has a s20,w value of 4.6 S indicating that the active fragment is a monomer of 65 kDa. Trypsin removes the first 77 amino acids which contain the aminoterminal dimerization site and the autophosphorylation sites. The Km and Vmax values of the fragment for ATP and Kemptide were essentially the same as those for the native enzyme. The fragment binds 2 mol cGMP/mol fragment with affinities close to that of the native enzyme. However, binding of cGMP to these sites was non-cooperative and shows similar characteristics to the autophosphorylated native enzyme. These results indicate that the aminoterminal dimerization site of cGMP-dependent protein kinase and the autophosphorylation site, present in this part, control not only the activation of the enzyme but also the cooperative binding characteristics of the intact enzyme.