A 28-d partial life-cycle test with the estuarine amphipod Leptocheirus plumulosus was developed in response to the need for an assay to mimic chronic exposure to sediment-associated contaminants. To ensure that toxicity tests have environmental relevance, it is essential to evaluate the relationship between laboratory responses and field measures of contamination. Consequently, one objective of the study was to compare the results of the chronic sediment toxicity test with L. plumulosus to gradients of sediment contamination and the in situ benthic community in its native Chesapeake Bay. Chronic tests were conducted by two laboratories, the Army Corps of Engineers Waterways Experiment Station ([WES]; Vicksburg, MS, USA) and the University of Maryland ([UM] College Park, MD, USA) using different feeding regimes, providing the opportunity to evaluate the effect of this variable on response sensitivity. A second objective was to compare the relative sensitivity of acute and chronic tests with L. plumulosus with field-collected sediments. Overall, there was good agreement between the toxicological response of acute and chronic tests with L. plumulosus and field measures of contamination. Survival in the acute test and chronic test conducted by WES was negatively correlated with concentrations of sediment-associated contaminants. Survival in acute exposures was significantly reduced in sediments from 8 of 11 stations. Indigenous L. plumulosus were found only at two of the three stations that did not exhibit acute toxicity. An unexpected finding was the difference in responsiveness of the two chronic tests. Survival in tests conducted by UM and WES was significantly reduced in sediments from 4 and 6 of 11 stations, respectively. No additional sublethal toxicity was detected in the UM chronic test, but the WES test detected reproductive effects at two additional stations. We believe the observed differences were related to the test diet used. Partly as a result of our findings, the recommended diet for the L. plumulosus chronic test was changed in the final methods document.