Genetically engineered crops expressing Bacillus thuringiensis (Bt) insecticidal crystalline (Cry) proteins became commercially available in the United States in 1996. In 2006, 19 million ha of Bt corn were planted worldwide, which represents a 10 million ha increase in 10 years. The sustainability of Bt crops is important, because their use has significantly reduced the amount of chemical insecticides necessary to control agricultural pests. Despite the high adoption rates of this novel insecticide, little is known about the aquatic fate of transgenic Bt proteins and their nontarget effects on aquatic invertebrates, although several potential routes exist for their transport to aquatic systems. Methods were developed to investigate the aquatic fate of transgenic Bt proteins and to determine their potential effects on nontarget aquatic invertebrates. Laboratory microcosms containing pond water only or pond water and sediment were used to examine the fate of the coleopteran-active Bt Cry3Bb1 protein in decomposing transgenic corn event MON863 (hereafter referred to as MON863 corn) leaf, stalk, and root. A half-life of less than 3 d was found for Bt Cry3Bb1 from decomposing MON863 corn residue. No Bt Cry3Bb1 was measured in the pond water or sediment extracts of microcosms containing MON863 corn. In an acute, static, partial-renewal toxicity test, Bt Cry3Bb1 protein from MON863 root extracts was fed to Chironomus dilutus larvae for 10 d. A significant decrease in C. dilutus survival at nominal concentrations of 30 ng/ml was found; however, no effect on growth among the surviving larvae was observed.