In the absence of cyclic nucleotides, the cAMP-dependent protein kinase and cGMP-dependent protein kinases (cGKs) suppress phosphotransfer activity at the catalytic cleft by competitive inhibition of substrate binding with a pseudosubstrate sequence within the holoenzyme. The magnitude of inhibition can be diminished by autophosphorylation near this pseudosubstrate sequence. Activation of type I cGK (cGKI) and type II cGK (cGKII) are differentially regulated by their cyclic nucleotide-binding sites. To address the possibility that the distinct activation mechanisms of cGKII and cGKI result from differences in the autophosphorylation of the inhibitory domain, we investigated the effects of autophosphorylation on the kinetics of activation. Unlike the type I cGKs (cGKIalpha and Ibeta), cGKII autophosphorylation did not alter the basal activity, nor the sensitivity of the enzyme to cyclic nucleotide activation. To determine residues responsible for autoinhibition of cGKII, Ala was substituted for basic residues (Lys(122), Arg(118), and Arg(119)) or a hydrophobic residue (Val(125)) within the putative pseudosubstrate domain of cGKII. The integrity of these residues was essential for full cGKII autoinhibition. Furthermore, a cGKII truncation mutant containing this autoinhibitory region demonstrated a nanomolar IC(50) toward a constitutively active form of cGKII. Finally, we present evidence that the dominant negative properties of this truncation mutant are specific to cGKII when compared with cAMP-dependent protein kinase Calpha and cGKIbeta. These findings extend the known differences in the activation mechanisms among cGK isoforms and allow the design of an isoform-specific cGKII inhibitor.