Listeriolysin O (LLO) is a primary virulence determinant of Listeria monocytogenes, a Gram-positive, facultative intracellular bacterium, that can cause severe diseases such as meningitis, septicaemia and gastroenteritis. LLO belongs to the large family of cholesterol-dependent cytolysins and forms pores in the plasma membrane of various cell types in sublytic concentrations. Using the patch-clamp-technique on HEK293 cells, we found that LLO-formed pores are transient and switch from open to closed states with different kinetics. We examined, whether this is due to a toxin-dependent or to a cell-dependent mechanism. Closing events were markedly reduced in outside-out patches in comparison to whole-cell-measurements. In whole-cell-measurements with human erythrocytes, which have little endo-and exocytotic acitvity, closings occurred hardly ever. In HEK293 cells, closing events appeared more frequently after pretreatment with 20 mM Cytochalasin D. In contrast, closures were strongly inhibited by removal of intra- and extracellular Ca²⁺. Our data demonstrate, that LLO-formed pores are closed by a cellular, Ca²⁺-dependent repair mechanism. (Supported by the Graduiertenförderung der Justus-Liebig-Universität Giessen).