In the present study, we addressed the interactions between ultrasound, microbubbles and living cells as well as consequent arising bioeffects. We specifically investigated whether hydrogen peroxide (H₂O₂) is involved in transient permeabilization of cell membranes in vitro after ultrasound exposure at low diagnostic power, in the presence of stable oscillating microbubbles, by measuring the generation of H₂O₂ and Ca²⁺ influx. Ultrasound, in the absence or presence of SonoVue^tm microbubbles, was applied to H9c2 cells at 1.8 MHz with a mechanical index (MI) of 0.1 or MI 0.5 during ten seconds. This was repeated every minute for five times. The production of H₂O₂ was measured intracellular with CM-H₂DCFDA. Cell membrane permeability was assessed by measuring real-time changes in intracellular Ca²⁺ with Fluo-4 using live-cell fluorescence microscopy. Ultrasound, in the presence of microbubbles, caused a significant increase in intracellular H₂O₂ at MI 0.1 of 50% and at MI 0.5 of 110% compared to control (p<0.001). Furthermore, we found increases in intracellular Ca²⁺ at both MI 0.1 and 0.5 in the presence of microbubbles, which was not detected in the absence of extracellular Ca²⁺. In addition, in the presence of catalase Ca²⁺ influx immediately after ultrasound exposure was completely blocked at MI 0.1 (p<0.01) and reduced by 50% at MI 0.5 (p<0.001). Finally, cell viability was not significantly affected, not even 24 hours later. These results implicate a role for H₂O₂ in transient permeabilization of cell membranes induced by ultrasound-exposed microbubbles.