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 P4, P3, P2, P'1, and P'2 positions. Substrates with 6,2-ANSN at P'1 had the highest fluorescence quantum yield, exceeding that of 6,1-ANSN 9.3-fold and 5,1-ANSN almost 1000-fold. Almost 50 substrates containing substituted ANSNs as leaving groups have KM values for APC below 100 microM, reaching as low as 4-10 microM for 8 of these substrates. These values are significantly lower than those reported for p-nitroanilide and 4-methylcoumaryl-7-amide substrates. Additionally, some of these substrates have relatively high kcat exceeding 50 s-1. These constants as well as kcat/KM are influenced by the nature of amino acid in the P3 and P2 positions, by the isomer of ANSN (P'1), and by the structure of substituent incorporated in the sulfonamide moiety (P'2). The highest kcat/KM were found for substrates with D-isomers of Leu, Phe, and Val in the P3 position when these amino acids were N-unblocked. For the P2 position Val, Phe, and Leu were preferable. Substrates containing n-butyl (bovine APC) and benzyl (human APC) substituents in the (P'2) structure have elevated kcat/KM. ANSN-containing substrates are hydrolzyed by both human and bovine APC at a similar rate.