In vitro extraction of sediments using digestive fluid of deposit-feeding invertebrates has been advocated as a measure of in vivo bioavailability, but the standard procedure used for the extractions alters fluid properties relative to in vivo conditions. In vivo fluid properties (redox potential [Eh], pH, dissolved oxygen) were measured in the gut of the polychaete Arenicola brasiliensis, and then the effects of gut evacuation, elevated temperature, and atmospheric exposure on these properties and/or metal and metalloid extraction capability of the fluid was assessed.The midgut of A. brasiliensis has an electropositive Eh of about +170 to +230 mV, a neutral pH, and little or no dissolved oxygen. Exposure to the atmosphere during in vitro extraction increased gut fluid oxygen content and affected the ability of the fluid to extract some trace elements from sediment particles, particularly Pb, Ni, Cu, Mn, and As. Even for these substances, however, the effects of oxygen exposure on extractability by gut fluid typically were small (<2-fold difference in amount extracted). For Cr, Hg, Se, Cd, Fe, and Zn, the effects of oxygen exposure usually were minor and inconsistent in direction. Maintaining anaerobic conditions during in vitro contaminant extractions is necessary when the experimental objective is to mimic accurately in vivo conditions and predict trace element extraction in the gut. If using metal extractability as a relative measure for sediment risk assessment purposes, a high degree of accuracy may not be required, and aerobic in vitro extractions could be suitable and produce only minor errors in extractability relative to in vivo conditions.