Escherichia coli -hemolysin (HlyA), archetype of a bacterial pore-forming toxin, has been reported to deregulate physiological Ca²⁺ channels, thus inducing periodic low-frequency Ca²⁺ oscillations that trigger transcriptional processes in mammalian cells. The present study was undertaken to delineate the mechanisms underlying the Ca²⁺ oscillations. Patch-clamp experiments were combined with single cell measurements of intracellular Ca²⁺ and with flowcytometric analyses. Application of HlyA at subcytocidal concentrations provoked Ca²⁺ oscillations in human renal and endothelial cells. However, contrary to the previous report, the phenomenon could not be inhibited by the Ca²⁺ channel blocker nifedipine and Ca²⁺ oscillations showed no constant periodicity at all. Ca²⁺ oscillations were dependent on the pore-forming activity of HlyA: application of a nonhemolytic but bindable toxin had no effect. Washout experiments revealed that Ca²⁺ oscillations could not be maintained in the absence of toxin in the medium. Analogously, propidium iodide flux into cells occurred in the presence of HlyA, but cells rapidly became impermeable toward the dye after toxin washout, indicating resealing or removal of the membrane lesions. Finally, patch-clamp experiments revealed temporal congruence between pore formation and Ca²⁺ influx. We conclude that the nonperiodic Ca²⁺ oscillations induced by HlyA are not due to deregulation of physiological Ca²⁺ channels but derive from pulsed influxes of Ca²⁺ as a consequence of formation and rapid closure of HlyA pores in mammalian cell membranes.--Koschinski, A., Repp, H., Ünver, B., Dreyer, F., Brockmeier, D., Valeva, A., Bhakdi, S., and Walev, I. Why Escherichia coli -hemolysin induces calcium oscillations in mammalian cells -the pore is on its own.