Calmodulin antagonists such as N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), which bind to calmodulin (CaM) in the presence of Ca2+ and selectively inhibit CaM-induced enzyme activation, contain a hydrophobic moiety. In this study, the naphthalenesulfonamide derivatives that lacked the chlorine molecule were less hydrophobic than those with chlorine. The chlorine-deficient derivatives also were less able to suppress the fluorescence of the hydrophobic probe (2-p-toluidinylnaphthalene-6-sulfonate) in the presence of the Ca2+-CaM complex. The affinity of naphthalenesulfonamides for Ca2+-CaM correlated well with their hydrophobicity and their potency in inhibiting Ca2+-CaM-dependent enzymes such as Ca2+-dependent cyclic nucleotide phosphodiesterase. The correlation between their hydrophobicity and affinity for the Ca2+-CaM complex also was observed when derivatives with various lengths of alkyl chain were used and when bromine, fluorine, or cyanogen was substituted for chlorine. Our observations suggest that these CaM antagonists may bind to the Ca2+-CaM complex through a hydrophobic interaction.