Troponin I inhibited, concentration-dependently, [3H]-N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and [3H]-trifluoperazine (TFP) binding to purified bovine brain calmodulin (CaM). Selective oxidation of methionine residues of CaM by N-chlorosuccinimide resulted in a rapid decrease in [3H]-W-7, [3H]-TFP and [14C]-chlorpromazine binding concomitant with the loss of CaM activity. Carbethoxylation of histidine residues, nitration of tyrosine residues and chemical modification of arginine residues with 1,2-cyclohexanedione produced no significant changes either in [3H]-W-7 binding to CaM or in the ability of CaM to stimulate phosphodiesterase. Our results suggest that the binding sites of these CaM antagonists on CaM may be located between the second and third Ca2+-binding loops.