Hyaluronidases are enzymes controlling many crucial physiological processes. Imbalanced enzymatic activity is connected with severe diseases. Because there is limited availability of drugs modulating hyaluronidase activity, the search for hyaluronidase interacting compounds is getting more and more important. A series of fifteen indole carboxamides and acetamides were synthesized and tested on inhibition of bovine testes hyaluronidase. In vitro assays were performed using stains-all at pH 7 and the Morgan-Elson reaction at pH 3.5. At neutral pH, the most active inhibitory compound was N-(Pyridin-4yl)-[5-bromo-1-(4-fluorobenzyl)indole-3-yl]carboxamide (20) with an IC(50) value of 46 microM. Surprisingly, inhibition of all compounds was completely abolished by a decrease in pH. At pH 3.5 the activity of the enzyme was increased up to 134% by compound N-(4,6-Dimethylpyridin-2yl)-(1-ethylindole-3-yl)acetamide (24) at a concentration of 100 microM. The known activating effect of bovine serum albumine (BSA) on hyaluronidase activity was verified in the assay and compared to the effect of compound 24. Structure-activity relationships are discussed and a model is proposed, which explains the increase in activity at pH 3.5 by bonding of the protonated form of N-(4,6-Dimethylpyridin-2yl)-(1-ethylindole-3-yl)acetamide (24) to hyaluronic acid. The bonding results in an elongated form of the substrate with easier enzymatic access.