A littoral enclosure protocol designed for aquatic field testing of pesticides is described. The enclosures are replicated, relatively economical, and incorporate all trophic levels, the natural shoreline, and undisturbed bottom sediments. The results of using this protocol to study the environmental chemistry of chlorpyrifos and the effects of chlorpyrifos on the biota in the enclosures are discussed. Chlorpyrifos degradation curves within treatment levels are similar; there was a rapid decline in concentration 1 h after treatment and a slower-than-expected vertical mixing rate occurred. Invertebrate population responses to chlorpyrifos application were generally more severe than would be expected based on laboratory LC₅₀ information. Fathead minnow (Pimephales promelas) and bluegill (Lepomis macrochirus) mortality were similar to predictions based on laboratory data. Significant reductions in growth rates of larval fish appear to be related to dietary changes caused by reductions in chlorpyrifos-sensitive invertebrate forage groups. The results show that this protocol will measure the dissipation, mixing, and persistence of a pesticide, provide information on primary and secondary (ecological) effects on invertebrate and fish populations, and allow a comparison of laboratory toxicity data to actual effects in a natural aquatic system.