D-Gluconic acid and alpha-carboxymethyl-polyethylene-glycol-omega-methyl ether (PEG) (mol wt 550) were covalently bound at N alpha-amino group of H-Phe-Arg-pNa to study the effect on hydrolysis by arginyl-hydrolases of chromogenic substrates containing high hydrophilic and amphiphilic groups. For comparison, epsilon-aminocaproyl-, sarcosyl- and succinyl-Phe-Arg-pNa were also synthesized. The obtained compounds were assayed as substrates for porcine pancreatic kallikrein, horse urinary kallikrein, tonin and beta-trypsin. Both PEG- and gluconyl-Phe-Arg-pNa had kcat values of hydrolysis 2-4 times higher than the N-acetyl derivative for all the studied enzymes. epsilon-NH2caproyl-Phe-Arg-pNa resulted in the best chromogenic substrate described for the two tissue kallikreins. The PEG-derivative and D-gluconyl groups were also introduced in the N alpha-amino group of H-Arg-pNa and assayed as beta-trypsin substrates. In comparison with benzoyl-Arg-pNa, the D-gluconyl group had no effect on Km but reduced the kcat value more than 15 times; however, PEG-Arg-pNa was hydrolyzed with similar Km but with kcat 5 times higher. The presence of D-gluconyl and PEG groups in the chromogenic substrate molecules increased their water solubility significantly.