Previous studies have shown that ultrasound (US) could enhance cellular uptake and cytotoxicity of chemotherapeutic agents in drug-resistant cancer cells. The goal of this study was to investigate the optimization of physical parameters of US exposure for in vitro reversal of multidrug resistance (MDR) in human hepatocarcinoma cell line (HepG2). Using a constant total energy density (3.87 J/cm(2)) that could maintain cell viability at the 90% level, we exposed parent (HepG2) and MDR variant (HepG2/ADM) tumor cells to US in vitro to a variety of US frequency, exposure intensity and duty cycle. After US exposure, flow cytometry was performed to measure retention of rhodamine 123 (Rh123) in both HepG2 and HepG2/MDR cells. The results showed that US frequency and duty cycle (DC) could influence the intracellular retention of Rh123 in HepG2/ADM tumor cells; intensity and exposure duration appeared to be of little importance. At a constant total energy density of 3.87 J/cm(2), the optimal US parameters for in vitro reversal of MDR in HepG2/ADM tumor cells appear to be 0.8 MHz, 0.43 W/cm(2) and 60% DC, respectively. These findings support our hypothesis that varying the physical parameters would have an effect on efficiency of US-mediated reversal of MDR in cancer cells.