Purpose: As basic studies to realize in vivo sonoporation, rates of cell membrane damage during sonoporation were evaluated using monolayer cells cultured on scaffolds withdifferent degrees of stiffness. Methods: Four types of scaffolds, constructed using collagen gel, 10 and 30% acrylamide gels, and a coverslip, were used for cultivation of monolayer cells. Young’s moduli measured using an atomic force microscope were in the range 0.09-8.6 kPa for the gel scaffolds, whereas Young’s modulus for living cells was 4.5 kPa. Cells with attached microbubbles were exposed to one-shot pulsed ultrasound of 8.0/-1.3 MPa in peak positive/negative pressures with durations of 3, 100, and 10,000 cycles. Results: Cell membrane damage was visualized by fluorescence microscopy using propidium iodide. The 3-cycle ultrasound pulse had no significant effect; however, the rates of damage caused by 100-cycle and 10,000-cycle pulses showed a strong tendency for higher rates of damage with a higher Young’s modulus. Conclusion: The experimental results indicate that the stiffness of the underlying layer of adherent cells should be considered as an essential parameter of the sonoporation condition and that the optimum exposure conditions for in vivo sonoporation should be determined with consideration of the physical properties of underlying tissues.