Piperonyl butoxide (PBO) is a synergist of pyrethroid pesticides found in many products for structural pest control, mosquito control, and home and garden uses. Because both PBO and pyrethroid residues potentially co-occur in urban creeks, this study determined if environmental levels of PBO were capable of synergizing pyrethroids in the environment. Three types of toxicity tests were conducted with the amphipod Hyalella azteca to determine the minimum PBO concentration required to increase toxicity of the pyrethroid permethrin: Sediment was spiked with permethrin only; permethrin and overlying water spiked with PBO; and permethrin, PBO, and overlying water spiked with PBO. In tests with PBO added to both water and sediment, PBO concentrations of 2.3 μg/L in water and 12.5 μg/kg in sediment reduced the permethrin median lethal concentration (LC50) nearly 50% to 7.3 mg/kg organic carbon (OC). Higher concentrations of PBO increased permethrin toxicity up to sevenfold. In exposures with PBO in water alone, 11.3 μg/L was required to increase permethrin toxicity. Urban creek sediments from California and Tennessee, USA, had PBO concentrations in the low μg/kg range; only one water sample was above the detection limit of 0.05 μg/L. Wetlands in northern California also were sampled after application of pyrethrins and PBO for mosquito abatement. Sediment and water PBO concentrations within 12 h of abatement spraying peaked at 3.27 μg/kg and 0.08 μg/L, respectively. These results suggest that environmental PBO concentrations rarely, if ever, reach concentrations needed to increase pyrethroid toxicity to sensitive organisms, though available data on environmental levels are very limited, and additional data are needed to assess definitively the risk.