Listerolysin O (LLO) is a major virulence factor of the bacterial human pathogen Listeria (L.) monocytogenes and is a member of the large family of cholesterol-dependent cytolysins (CDCs). In the facultative intracellular life cycle of L. monocytogenes, the ability of LLO to form pores is crucial for lysis of the host phagosome. Further to lysis, LLO induces multiple host cell responses in sublytic concentrations. Since thiols enhance hemolytic activity of CDCs, they formerly were referred to as thiol-activated cytolysins. This term was abolished some years ago, since classical hemolysis experiments neither enlightened relevance nor underlying mechanisms of thiol-activation. The aim of the present study was to test whether thiol activation is detectable with the patch clamp technique, which allows direct monitoring of pore formation. Indeed, we observed that L- cysteine or N-acetylcysteine enhanced formation of LLO pores about threefold with an EC50 of about 300 mM. This might be due to enhanced simultaneous openings of coupled pores or to formation of pores with higher conductances. Thus, thiol- activation appears as an important property for fine tuning of LLO activity, allowing specific exertion of lytic and non-lytic functions during the life cycle of L. monocytogenes. (Supported by Graduiertenförderung der Justus-Liebig-Universität Gießen)