Ammonia commonly is present at detectable concentrations in overlying and interstitial water in solid-phase sediment toxicity tests. There are scenarios in which it is desirable to discern ammonia toxicity from that caused by other contaminants. The objective of this study was to verify the technical basis for defining the bioavailability of ammonia in solid-phase sediment toxicity tests based on interstitial-(pore-) water concentrations of the compound. We compared the toxicity of ammonia in spiked-sediment versus water-only exposures with the oligochaete Lumbriculus variegatus, the midge Chironomus tentans, and the amphipod Hyalella azteca. Sediment exposures were conducted in a test system that enabled the maintenance of target concentrations of ammonia in pore water, while keeping overlying water concentrations of the compound below potential effect levels. To enable direct comparison of the water-only and sediment exposures, the same test conditions (including pH) were used for each. There was good correspondence between the LC50 values of water-only tests and spiked-sediment toxicity tests for both L. variegatus and C. tentans. Hyalella azteca apparently avoided the spiked sediments and was frequently observed in the less contaminated overlying water, thus limiting the evaluation of a pore-water exposure model for the amphipod. Overall, at least for some benthic species, ammonia bioavailability and toxicity can be accurately predicted from pore-water concentrations of the compound; however, the model maybe less robust for more epibenthic organisms, such as H. azteca.