A series of 87 fluorescent peptide substrates have been synthesized and evaluated using human and bovine activated protein (C) (APC). These substrates contain various isomers of aminonaphthalenesulfonamides (ANSN) as the detecting group and were substituted at (\mathbf{P}{4}, \mathbf{P}{3}, \mathbf{P}{2}, \mathbf{P}{1}^{\prime}), and (\mathbf{P}{2}^{\prime}) positions. Substrates with 6,2 ANSN at (P{1}^{\prime}) had the highest fluorescence quantum yield, exceeding that of 6,1-ANSN 9.3-fold and 5,1ANSN almost 1000-fold. Almost 50 substrates containing substituted ANSNs as leaving groups have (K_{M}) values for APC below (100 \mu \mathrm{M}), reaching as low as 4-10 (\mu) M for (\mathbf{8}) of these substrates. These values are significantly lower than those reported for (p)-nitroanilide and 4methylcoumaryl-7-amide substrates. Additionally, some of these substrates have relatively high (\boldsymbol{k}{\text {cat }}) exceeding (50 \mathrm{~s}^{-1}). These constants as well as (k{\text {cat }} / K_{M}) are influenced by the nature of amino acid in the (P_{3}) and (P_{2}) positions, by the isomer of ANSN (\left(P_{1}^{\prime}\right)), and by the structure of substituent incorporated in the sulfonamide moiety (\left(P_{2}^{\prime}\right)). The highest (\boldsymbol{k}{\mathrm{cat}} / \boldsymbol{K}{M}) were found for substrates with D-isomers of Leu, Phe, and Val in the (P_{3}) position when these amino acids were (\mathrm{N})-unblocked. For the (P_{2}) position Val, Phe, and Leu were preferable. Substrates containing n-butyl (bovine APC) and benzyl (human APC) substituents in the (\left(\mathbf{P}{\mathbf{2}}\right.) ) structure have elevated (\boldsymbol{k}{\text {cat }} / \boldsymbol{K}_{\mathrm{M}}). ANSN-containing substrates are hydrolyzed by both human and bovine APC at a similar rate. 1995 Academic Press, Inc.