A toxicity identification evaluation (TIE) conducted on a municipal wastewater discharge from the southeast United States was part of a research project aimed at developing U.S. Environmental Protection Agency (EPA) TIE methods for acutely toxic effluents. The effluent consistently exhibited acute toxicity to Ceriodaphnia dubia but not to fathead minnows (Pimephales promelas). Toxicity characterization procedures revealed that the primary toxicant was a nonpolar organic. Toxicity was recovered through C18 solid-phase extraction and concentration steps. Gas chromatography–mass spectroscopy of these concentrates revealed the presence of diazinon (O, O-diethyl O-[6-methyl-2-(1-methylethyl)-4-pyrimidinyl] phosphorothioate). Diazinon concentrations in whole effluent, determined by GC analyses, correlated well with the toxicity measurements of each sample. Relative species sensitivity also implicated diazinon as the primary toxicant. This study illustrates the successful application of EPA TIE methodologies for identifying a nonpolar organic toxicant in a complex effluent. The significance of detecting diazinon at acutely toxic concentrations in municipal wastewater may indicate a more widespread problem in this region of the United States. This toxicity problem may be attributed to the chemical characteristics of diazinon and its applications.