Road runoff is recognized as a substantial nonpoint source of contamination to the aquatic environment. Highway seasonal first flushes contain particularly high concentrations of pollutants. To fully account for the toxicity potential of the runoff, the cumulative effects of the pollutants should be assessed, ideally by biological analyses. Acute toxicity tests with Daphnia magna were used to measure the toxicity of runoff from three major highway sections in Israel for 2 yr. Highway first flushes resulted in the mortality of all tested individuals within 24 to 48 h. A first flush collected from Highway 4 (traffic volume: 81,200 cars d-1) remained toxic even after dilution to <5% (48 h EC50 <5%). Synthetic solutions with metal concentrations corresponding to highways' first flushes revealed a synergistic adverse effect on D. magna survival and a potential additive effect of nonmetal pollutants in the runoff. Because daphnids and other invertebrates constitute the base of the aquatic food chain, detrimental effects of highway runoff may propagate to higher levels of biological organization. The observed high potential of environmental contamination warrants the control of highway runoff in proximity to natural watercourses.