Poultry litter is a mixture of excreta, feed, feathers, and bedding material. Poultry litter is useful as a fertilizer due to the presence of nitrogen, phosphorus, and potassium. Litter is commonly used for growing corn, soybeans, potatoes, tomatoes, leafy vegetables, and cover crops. The aqueous leachate of poultry litter has been shown to exhibit toxicity to a variety of indicator organisms. The objective of this research was to identify classes of toxicants in the litter aqueous leachate. The aqueous extract of poultry litter was subjected to toxicity identification tests approved by the United States Environmental Protection Agency that use Ceriodaphnia dubia as the test organism. Tests performed included pH adjustment, filtration, aeration, C18 solid-phase extraction, EDTA addition, sodium thiosulfate addition, and extraction through zeolite. Zeolite extraction, filtration at low pH, and aeration at low pH reduced overall toxicity by 68, 20, and 22%, respectively. The major sources of toxicity appear to be ammonia and anionic organic compounds. Toxicity was apparently not due to the presence of heavy metals or oxidants.