There is increasing public attention concerning the effect of endocrine disruptor chemicals (EDCs) on the immune system. One important group belonging to EDCs are the environmental estrogens. Commonly found in the effluents in wastewater treatment plants, 17α-ethynylestradiol (EE2) which is used in contraceptive pills, is an endocrine disruptor with strong estrogenic effects. This study aims to investigate the capacity of EE2 to modulate in vivo and in vitro the innate immune response of the gilthead seabream (Sparus aurata L.), a teleost species of great commercial value. For this purpose, adult specimens were bath-exposed to EE2 (0, 5 and 50 ng/L) and then immunized with hemocyanin in the presence of the adjuvant aluminum. The results indicate that, after 15 days of EE2-exposure, the disruptor was able to inhibit in a dose-dependent manner the induction of interleukin-1β (IL-1β) gene expression, but did not significantly alter the specific antibody titer. To shed light on the role played by EE2 into seabream immune response, leukocytes were exposed in vitro to several concentrations of EE2 (0, 0.5, 5, 50 and 500 ng/ml) for 3, 16 and 48 h and the production of reactive oxygen intermediates, the phagocytic activity and the gene expression profile of these cells were analyzed. EE2 was seen to inhibit both cellular activities and to alter the immune gene expression profile in primary macrophages. Thus, low concentrations of EE2 increase the mRNA levels of IL-1β, IL-6, tumour necrosis factor α and tumour growth factor β in non-activated macrophages. In contrast, EE2 treatment of activated macrophages resulted in the decreased expression of pro-inflammatory genes and the increased expression of genes encoding anti-inflammatory and tissue remodeling/repair enzymes. Taken together, our results suggest that EE2 might alter the capacity of fish to appropriately respond to infection although it does not behave as an immunosuppressor.