Macrophage aggregates (MAs) are structures in the spleen, kidney and sometimes liver of fishes which have various functions such as recycling/storing/detoxification of cellular wastes and exogenous substances. They have been also reported to be important in the specific immune response and are used as health indicators. Changes in MA density, size and pigment content have been used in national and local monitoring programs in the U.S. as indicators of contaminant exposure. However, MA number and structure can also be affected by other factors, including general stress or nutritional status of fish. An experiment was conducted to study the effects of vitamin E and n−3 highly unsaturated fatty acid (HUFA) deficiencies and stocking density on spleen and kidney MAs of gilthead seabream, one of the most important species for Mediterranean aquaculture. Fish were held at two stocking densities, high and low, and fed experimental diets. Diet NE had no supplemental vitamin E, Diet NFA was deficient in n−3 HUFA and Diet C was a control diet. Number, size and shape factor of MAs were measured using image analysis. The percentage of tissue occupied by MAs was calculated from these measurements. The results showed that high stocking density alone increased the number of splenic but not kidney MAs of fish fed the control diet. A deficiency of n−3 HUFA alone also increased the number of splenic but not kidney MAs at both stocking densities. Vitamin E deficiency alone had no significant effect on MAs in either organ. However, the combined effect of vitamin E deficiency and high stocking density increased the number and size of kidney but not splenic MAs. This study indicates that specific dietary deficiencies can influence MA accumulation and that splenic MAs may be more responsive to general stress than kidney MAs.