Del Puerto Creek, an agriculturally influenced stream in northern California, USA, with a history of sediment toxicity, was used as a case study to determine the feasibility of using sediment toxicity testing and chemical analysis to identify the causative agent for the toxicity and its sources. Testing with the amphipod Hyalella azteca confirmed historical toxicity and identified a point along the creek at which there was an abrupt increase in sediment toxicity that persisted for at least 6 km downstream. Three recently developed whole sediment toxicity identification evaluation manipulations, temperature reduction, piperonyl butoxide addition, and esterase addition, were applied to sediment from one site and were suggestive of a pyrethroid as the cause for toxicity. Utilizing published median lethal concentration (LC50) values in a toxic unit analysis, the pyrethroid insecticide bifenthrin was identified as the primary contributor to toxicity in nearly all sites at which toxicity was observed, with occasional additional contributions from the pyrethroids lambda-cyhalothrin, esfenvalerate, and cyfluthrin. Most agricultural drains discharging to Del Puerto Creek contained bifenthrin in their sediments at concentrations near or above acutely toxic concentrations. However, only one drain contained sediments with bifenthrin concentrations approaching the concentrations measured in creek sediments. This fact, along with the proximity of that particular discharge to the location in the creek with the highest concentrations, suggested that one drain may be responsible for much of the toxicity and pyrethroid residues in creek sediments. The methods employed in this study are likely to be of considerable value in total maximum daily load efforts in Del Puerto Creek or other California surface water bodies known to have pyrethroid-related aquatic toxicity.