Lysosomes are recruited to the invasion site during host cell entry by Trypanosoma cruzi, an unusual process suggestive of the triggering of signal transduction mechanisms. Previous studies showed that trypomastigotes, but not the noninfective epimastigotes, contain a proteolytically generated trypomastigote factor (PGTF) that induces intracellular free Ca2+ transients in several mammalian cell types. Using confocal time-lapse imaging of normal rat kidney (NRK) fibroblasts loaded with the Ca2+-sensitive dye fluo-3, we show that the initial intracellular free Ca2+ concentration ([Ca2+](i)) transient detected a few seconds after exposure to trypomastigote extracts is a result of Ca2+ release from intracellular stores. Removal of Ca2+ from the extracellular medium or inhibition of Ca2+ channels with NiCl2 did not affect the response to PGTF, while depletion of intracellular stores with thapsigargin abolished it. [Ca2+](i) transients induced by PGTF were shown to be coupled to the activity of phospholipase C (PLC), since the specific inhibitor U73122 completely blocked the response, while its inactive analogue U73343 had no effect. In addition, polyphosphoinositide hydrolysis and inositol 1,4,5-trisphosphate (IP3) were detected upon cell stimulation with PGTF, suggesting the participation of IP3-sensitive intracellular Ca2+ channels. An immediate effect of the signaling induced by PGTF and live trypomastigotes was a rapid and transient reorganization of host cell microfilaments. The redistribution of F-actin appeared to be a direct consequence of increased [Ca2+](i), since thrombin and the Ca2+ ionophore ionomycin produced a similar effect, with a time course that corresponded to the kinetics of the elevation in [Ca2+](i). These observations support the hypothesis that PGTF-induced disassembly of the cortical actin cytoskeleton may play a role in T. cruzi invasion, by facilitating lysosome access to the invasion site. Taken together, our findings suggest that the proteolytically generated trypomastigote factor PGTF is a novel agonist that acts through the PLC/phosphoinositide signaling pathway of mammalian cells.