The effect of alteration of the aromatic hydrocarbon moicty of aromatic amidines on their inhibitory effects on trypsin [EC3.4.4.4] were investigated. δ-Naphthamidine (K, =6.6× 10−6M) was slightly more inhibitory than benzamidine, suggesting that the former interacts more effectively with the “hydrophobic site” of the enzyme. In sharp contrast, however, α-napthamidine was distinctly less inhibitory (Ki=2.4 × 10−4) than the β-derivative. This difference is interpreted in terms of the hydriphobic part of the inhibitor molecules. The binding of α- and β-naphthylmethylamines were similar. In addition to kinetic assays, the circular dichroic spectra were examined and gel filtration techniques were employed to examine the interaction between trypsin and β-naphthamidine.