Nanodroplet-mediated histotripsy (NMH) is a targeted ultrasound ablation technique combining histotripsy with nanodroplets that can be selectively delivered to tumor cells for targeted tumor ablation. In a previous study, it was reported that by use of extremely short, high-pressure pulses, histotripsy cavitation bubbles were generated in regions containing nanodroplets at significantly lower pressure (similar to 10.8 MPa) than without nanodroplets (similar to 28 MPa) at 500 kHz. Furthermore, it was hypothesized that lower frequency would improve the effectiveness of NMH by increasing the size of the focal region, increasing bubble expansion, and decreasing the cavitation threshold. In this study, we investigated the effects of ultrasound frequency (345 kHz, 500 kHz, 1.5 MHz, and 3 MHz) on NMH. First, the NMH cavitation threshold was measured in tissue phantoms with and without nanodroplets, with results indicating that the NMH threshold was significantly below the histotripsy intrinsic threshold at all frequencies. Results also indicated that the NMH threshold decreased at lower frequency, ranging from 7.4 MPa at 345 kHz to 13.2 MPa at 3 MHz. In the second part of this study, the effects of frequency on NMH bubble expansion were investigated, with results indicating larger expansion at lower frequency, even at a lower pressure. In the final part of this study, the ability of perfluoropentane-encapsulated nanodroplets to act as sustainable cavitation nuclei over multiple pulses was investigated, with results indicating that the nanodroplets are destroyed by the cavitation process and only function as cavitation nuclei for the first few pulses, with this effect being most pronounced at higher frequencies. Overall, the results of this study support our hypothesis that using a lower frequency will improve the effectiveness of NMH by increasing the size of the focal region, increasing bubble expansion and decreasing the cavitation threshold. (E-mail: email@example.com) (C) 2015 World Federation for Ultrasound in Medicine & Biology.