Abstract Ultrasound treatment has been shown to enhance the uptake of both hydrophilic and hydrophobic compounds into PC3 and Huvec cell lines using an insonation regimen of a single 10-s burst of high-frequency (4 MHz), moderate intensity (32 W/cm2) ultrasound. The purpose of this work was to evaluate the effect of this ultrasound regimen on the cellular accumulation of paclitaxel (PTX) loaded in copolymer micellar of methoxy poly(ethylene glycol)-block-poly(D,L-lactide) (MePEG-b-PDLLA) in both drug-sensitive (MDCKII and MCF-7) and P-glycoprotein (Pgp)–expressing (MDCKII-MDR and NCI-ADR) cell lines. There were no effects of ultrasound on hydrodynamic diameters of micelles and the release of FRET pairs, indicating the integrity of micelles was maintained. There was a two-fold increase in intracellular PTX for all ultrasound-treated drug-sensitive cell lines and their respective drug-resistant counterparts compared with no ultrasound. Significant decreases in drug efflux rates were observed at 20, 40 and 60 min for both drug-sensitive and -resistant cell lines receiving ultrasound. The enhanced accumulation and retention of PTX by ultrasound resulted in greater cytotoxicity in both MDCKII and MDCKII-MDR cell lines, as indicated by the MTS assay. These data suggest that ultrasound may facilitate the uptake of intact paclitaxel-loaded micelles into cells, allowing greater retention of drug in both Pgp and non-Pgp–expressing cells.