Previous studies have shown that if a solution for cell suspension is saturated with CO(2), ultrasound-induced in vitro cell killing and free-radical production are inhibited. However, the dose dependency of this observation has not been explored. Here, we used NaHCO(3) and HCl to produce a predictable concentration of CO(2) within the culture medium. Using 1 MHz continuous wave 4 W/cm(2) ultrasound, we sonicated U937 cells suspension for 1 min at 37 degrees C with CO(2) at different concentrations. At 2 mM, reduced cell killing was observed that further decreased with increasing CO(2) concentration until 100% protection was attained at 20 mM. Ultrasound-induced free-radical production was significantly decreased at 1 mM and became undetectable at 2 mM CO(2). This finding shows that CO(2)-mediated inhibition is concentration dependent and that the threshold for free-radical production is one order of magnitude higher than the threshold for cell killing induced by ultrasound. In addition, it also cautions researchers when adding acids and acid-based agents to a culture medium, which almost always contains NaHCO(3), in experiments related to the bioeffects of ultrasound.