The gastric lumen represents a bactericidal barrier, whose major components are an acidic pH and a family of isoenzymes of the gastric aspartate protease, pepsin. To evaluate whether specific pepsins are specialized in antibacterial protection, we tested their effects on the gastric pathogen Helicobacter pylori. In a recent study we found pepsin to affect the motility of the bacteria, one of its most important virulence factors. We were able to show that the antibacterial effect of pepsin occurs in two phases: rapid loss of motility and subsequent destruction. In the present study we used the rapid pepsin-induced bacterial immobilization as a marker of antibacterial efficiency. The proteolytic activity of different pepsins was normalized to values between 2 and 200 U/ml in the hemoglobin degradation test of Anson, performed at pH 2 and 5. We found that pepsin C completely inactivates H. pylori at proteolytic activities of 2 (pH 5) and 20 (pH 2) U/ml. In contrast, the activities of pepsin A and chymosin required to affect Helicobacter motility were ten times higher.