Recently, it has been demonstrated that the biocide tributyltin (TBT) can interfere with fish sex differentiation, leading to a bias of sex toward males. On the contrary, it is well known that estrogenic compounds can induce fish feminization. Yet, the combined effects of mixtures of androgenic and estrogenic compounds on fish sex differentiation have never been investigated before, even though in the environment animals are frequently exposed to both groups of xenobiotics. Therefore, in order to investigate whether exposure to estrogenic compounds can block the masculinizing effect of TBT, 5 days post-fertilization zebrafish (Danio rerio) larvae were exposed for a four month period to TBT and to the synthetic estrogen-ethinylestradiol (EE2). The fish were fed a diet containing TBT at nominal concentrations of 25 and 100 ng TBT/g, and two groups of animals were also dosed with TBT plus EE2 at nominal water concentration of 3.5 ng/L, using a flow-through design. As expected, fish exposed to TBT showed a bias of sex toward males (62.5% males in control tanks and 86% and 82% in TBT 25 and TBT 100 ng TBT/g, respectively). Co-exposure to EE2 completely blocked the masculinizing effect of TBT, with 7% males in the TBT 25 ng/g + EE2 treatment and 0% in the EE2 alone and in the TBT 100 ng/ + EE2 exposed groups. These results clearly indicate that EE2, at environmentally relevant concentrations, can block the TBT masculinizing effects in zebrafish, which suggests that in the aquatic environment the presence of estrogens may neutralize the fish masculinizing effect of TBT. Our findings highlight the need of testing the combined effects of contaminants, as single exposure studies may not be sufficient to predict the effects of mixtures of xenobiotics with antagonistic properties.