The cationic antimicrobial immunomodulatory peptide, KLK (KLKL5KLK), exerts profound membrane interacting properties, impacting on ultrastructure and fluidity. KLK-membrane interactions that lead to these alterations require the ability of the peptide to move into an a-helical conformation. We show that KLK induces an increase of the intracellular Ca2+ concentration in human T24 cells. The effect of KLK is buffer-sensitive, as it is detected when HBSS buffer is used, but not with PBS. This, together with the lack of effect of the middle leucine-to-proline-substituted peptide derivative [KPK (KLKLLPLLKLK)], indicates that it is the conformational propensity rather than the net positive charge that contributes to the effect of KLK on intracellular Ca2+ level of T24 cells. We show that, although KLK slightly stimulates Ca2+ influx into the cell, the bulk increase of Ca2+ levels is due to KLK-induced depletion of intracellular Ca2+ stores. Finally, we demonstrate a KLK- induced switch of PS (phosphatidylserine) from the inner to the outer plasma membrane leaflet that contributes to the onset of early apoptotic changes in these cells.