Cysteine protease-inhibiting proteins of the cystatin superfamily can inhibit the replication of certain viruses and bacteria. The inhibitory centre of human cystatin C, the most widely distributed human cystatin, comprises three peptide segments. The present work describes the synthesis and antibacterial activity of 27 new peptidyl derivatives or cyclopeptides based upon the aminoterminal segment Arg8-Leu9-Val10-Gly11. Fourteen of the new compounds displayed antibacterial activity against from 1 up to 9 of 17 clinically important bacterial species tested. Antiproteolytic activity of a compound was usually not required for its antibacterial capacity. Peptidyl diazomethanes generally had a very narrow antibacterial spectrum, inhibiting only Streptococcus pyogenes, whereas cyclopeptides and peptidyl derivatives of the general structure X-Arg-Leu-NH-CH(iPr)-CH2-NH-Y had a much wider spectrum. The most potent of these substances displayed approximately equal minimal inhibitory and bactericidal concentrations of about 20 microg/ml for both Staphylococcus aureus and S. pyogenes and were devoid of antiproteolytic activity. Several of the new substances could protect mice against lethal intraperitoneal challenge with S. pyogenes. Though their target remains to be disclosed, the group of substances here reported might be promising for the development of antibacterial drugs and the discovery of novel principles of action.