Because of the physiologic importance of hyaluronidases, the identification of potent and selective inhibitors of hyaluronidases has become increasingly important. A variety of assay methods have been used for such a purpose, i.e. classical turbidimetric, viscometric and colorimetric. In this study, a modified enzymatic assay has been used to obtain a microtiter plate-based sensitive activity screening. All inhibitors were tested in a stains-all assay at pH 7 and in a Morgan-Elson assay at pH 3.5. Among the tested compounds, 1, 2, 3, 6, 7, 8, 16, 17 and 18 showed good inhibition of more than 50%, so the IC(50) values of these derivatives were determined in the range of 25-41 microm. The IC(50) value of the most active hyaluronidase inhibitor Vcpal (6-palmitoyl-L-ascorbic acid) was measured as 8.36 microm. All inhibitors including Vcpal showed twofold less activity at pH 3.5 in a Morgan-Elson assay. Examination of substituent effects on the activity showed that para-positions of benzamide needs to be chlorinated or fluorinated to obtain good inhibitory effect. It was found that the introduction of a p-fluoro benzyl ring in the indole nitrogen has a positive effect for the inhibitory effects of both indole-2- and 3-carboxamide derivatives.