A series of new compounds, 6-amino-1-naphthalenesulfonamides (ANSN), were used as fluorescent detecting groups for substrates of amidases. These compounds have a high quantum fluorescent yield, and the sulfonyl moiety permits a large range of chemical modification. Fifteen ANSN substrates with the structure (N alpha-Z)Arg-ANSNR1R2 were synthesized and evaluated for their reactivity with 8 proteases involved in blood coagulation and fibrinolysis. Thrombin, activated protein C, and urokinase rapidly hydrolyzed substrates with monosubstituted sulfonamide moieties (R1 = H). The maximum rate of substrate homologue). The hydrolysis rates for substrates with branched substituents were slower than their linear analogues. Monosubstituted (N alpha-Z)Arg-ANSNR1R2 possessing cyclohexyl or benzyl groups in the sulfonamide moiety were hydrolyzed by these three enzymes at rates similar to that of the n-butyl homologue (except the cyclohexyl compound for u-PA). Factor Xa rapidly hydrolyzed substrates with short alkyl chains, especially when R1 = R2 = CH3 or C2H5. Lys-plasmin and rt-PA demonstrated low activity with these compounds, and the best results were accomplished for monosubstituted compounds when R2 = benzyl (for both enzymes). Factor VIIa and factor IXa beta exhibited no activity with these substrates. A series of 14 peptidyl ANSN substrates were synthesized, and their reactivity for the same 8 enzymes was evaluated. Thrombin, factor Xa, APC, and Lys-plasmin hydrolyzed all of the substrates investigated. Urokinase, rt-PA, and factor IXa beta exhibited reactivity with a more limited group of substrates, and factor VIIa hydrolyzed only one compound (MesD-LGR-ANSN(C2H5)2). The substrate ZGGRR-ANSNH (cyclo-C6H11) showed considerable specificity for APC in comparison with other enzymes (kcat/KM = 19,300 M-1 s-1 for APC, 1560 for factor IIa, and 180 for factor Xa). This kinetic advantage in substrate hydrolysis was utilized to evaluate the activation of protein C by thrombin in a continuous assay format. Substrate (D-LPR-ANSNHC3H7) was used to evaluate factor IX activation by the factor VIIa/tissue factor enzymatic complex in a discontinuous assay. A comparison between the commercially available substrate chromozyme TH (p-nitroanilide) and the ANSN substrate with the same peptide sequence (TosGPR) demonstrated that aminonaphthalenesulfonamide increased the specificity (kcat/KM) of substrate hydrolysis by thrombin more than 30 times, with respect to factor Xa substrate hydrolysis.