This study was undertaken to examine ultrasound (US) mechanisms and their impact on chemical and biological effects in vitro as a function of changing pulse repetition frequency (PRF) from 0.5 to 100Hz using a 1MHz-generator at low-intensities and 50% duty factor (DF). The presence of inertial cavitation was detected by electron paramagnetic resonance (EPR) spin-trapping of hydroxyl radicals resulting from sonolysis of water. Non-cavitational effects were evaluated by studying the extent of sucrose hydrolysis measured by UV spectrophotometry. Biological effects were assessed by measuring the extent of cell killing and apoptosis induction in U937 cells using Trypan blue dye exclusion test and flow cytometry, respectively. The results indicate significant PRF dependence with respect to hydroxyl radical formation, cell killing and apoptosis induction. The lowest free radical formation and cell killing and the highest cell viability were found at 5Hz (100ms pulse duration). On the other hand, no correlation was found between sucrose hydrolysis and PRF. To our knowledge, this is the first report to be devoted to study the impact of low PRFs at low-intensities on US-induced chemical and biological effects and the mechanisms involved. This study has introduced the role of "US streaming" (convection); a forgotten factor in optimization studies, and explored its importance in comparison to standing waves.