An esterase enzyme capable of catalyzing the hydrolysis of the ester bond common to all pyrethroid insecticides has been proposed as a toxicity identification evaluation procedure for the compounds when present in water samples. It appeared to show promise in previous applications to pore water and in one published application to bulk sediment. The present study was designed to provide the additional validation of the technique when applied to whole sediment, demonstrating its efficacy and specificity to pyrethroids. When added to the overlying water in a standard sediment toxicity test with the amphipod Hyalella azteca, the enzyme reduced toxicity of pyrethroid-spiked laboratory sediments. The technique had no effect on toxicity of DDT or chlorpyrifos, but it dramatically reduced the toxicity of cadmium, presumably by complexation of the cadmium with dissolved organic matter rather than by enzymatic activity. Esterase addition consistently reduced pyrethroid-related toxicity in 12 tests with field-collected sediments. The procedure, however, also results in substantial changes in water quality, provides an undesirable nutritional benefit to the test organisms exposed to the esterase, and achieves a substantial fraction of the protective capability simply by complexation of the pyrethroids with the esterase or the microbial growth that the esterase promotes. The esterase addition technique has potential for whole-sediment application, but further development of procedures and confirmation by independent lines of evidence are needed to reduce the potential for misinterpretation of results.