Acute toxicity and immune response, combined with temperature stress effects, were evaluated in larval and juvenile American lobsters (Homarus americanus) exposed to malathion, resmethrin and methoprene. These pesticides were used to control West Nile virus in New York in 1999, the same year the American lobster population collapsed in western Long Island Sound (LIS). Whereas the suite of pesticides used for mosquito control changed in subsequent years, a field study was also conducted to determine pesticide concentrations in surface waters on Long Island and in LIS after operational applications. The commercial formulations used in 2002 and 2003—Scourge, Anvil and Altosid—contain the active ingredients resmethrin, sumithrin and methoprene, respectively. Concentrations of the synergist piperonyl butoxide (PBO) were also measured as a proxy for pesticide exposure. Acute mortality in Stage I-II larval lobsters demonstrated that they are extremely sensitive to continuous resmethrin exposure. Resmethrin LC50s for larval lobsters determined under flow-through conditions varied from 0.26–0.95 μg L⁻¹ in 48- and 96-h experiments at 16°C, respectively. Increased temperature (24°C) did not significantly alter resmethrin toxicity. Malathion and methoprene were less toxic than resmethrin. The 48-h LC50 for malathion was 3.7 μg L⁻¹ and methoprene showed no toxicity at the highest (10 μg L⁻¹) concentration tested. Phenoloxidase activity was used as a measure of immune response for juvenile lobsters exposed to sublethal pesticide concentrations. In continuous exposures to sublethal doses of resmethrin (0.03 μg L⁻¹) or malathion (1 μg L⁻¹) for 7 d at 16 or 22°C, temperature had a significant effect on phenoloxidase activity (P ≤ 0.006) whereas pesticide exposure did not (P = 0.880). The analytical methods developed using high performance liquid chromatography coupled to time-of-flight mass spectroscopy (LC-TOF-MS) provided high sensitivity with mass detection limits of 0.1–0.3 ng L⁻¹. Pesticide levels were often detected in the ng L⁻¹ range in Long Island surface waters and western LIS (except in open waters), but rarely at concentrations found to be toxic in flow-through laboratory exposures, even immediately after spray events.