The utility of splenic macrophage aggregates (MAs) as an indicator of fish exposure to degraded environments was evaluated in several species of estuarine fishes as part of the Environmental Protection Agency's Environmental Monitoring and Assessment Program–Estuaries (EMAP-E). Using image analysis, we measured the number and mean size of MAs per square millimeter on tissue sections of spleen from 983 fishes representing seven species from 266 stations scattered across coastal estuaries of the Gulf of Mexico. At 16 stations, at least one fish exhibited a high density of MAs (>40 MAs/mm²). Densities of MAs that exceeded 40/mm² correlated with exposure to either hypoxic conditions or sediment contamination. Fisher's exact test showed that the observed frequencies of joint occurrence between high numbers of MAs and both high sediment contaminants and low dissolved oxygen were significantly greater than the expected background frequencies. For all 16 sites where MAs were greater than 40/mm², sediments displayed at least one contaminant at a concentration in the highest 5% of those observed for all Gulf of Mexico stations. Additionally, comparison of subjective visual analyses with the image analysis measurements showed a strong correlation, indicating that similar analyses can be performed without computer image analysis. This study demonstrates that splenic MAs are effective biotic indicators for discriminating between fish exposed to degraded and nondegraded environments.