The cysteine residues of cAMP-dependent protein kinase II from porcine heart have been probed using alkylation with iodoacetic acid. Alkylation of the dissociated catalytic subunit resulted in loss of activity that was concomitant with the alkylation of both cysteine residues, Cys 199 and Cys 343. In contrast, no loss of activity was observed following alkylation of the holoenzyme. Isolation of the C-subunit following dissociation of the alkylated holoenzyme with cAMP revealed that Cys 343 was fully alkylated whereas Cys 199 was completely protected from chemical modification. These results establish that Cys 343 is not essential for enzymatic activity and, furthermore, indicate that aggregation of the C- and R-subunits selectively protects Cys 199. Alkylation of the R-subunit was also characterized both in the presence and absence of C-subunit. Cysteines 97, 124, and 326 were alkylated in R2 both in the presence and absence of cAMP. Of these 3 residues only Cys 97 was protected from alkylation in the holoenzyme. In the presence of high concentrations of iodoacetic acid, partial alkylation of the remaining 3 cysteine residues was observed, and this labeling was eliminated by cAMP suggesting that cAMP results in a general tightening of the protein conformation rather than the selective protection of any single cysteine residue.