The aim of the current research was to assess the possible influence of copper sulphate contamination on the antioxidant enzymatic defenses and lipid peroxidation (LPO) in the mosquitofish (Gambusia holbrooki). Quadruplicated lots of this fish were exposed to three increasing sub-lethal concentrations of Cu (0.10; 0.17 and 0.25mgCu/L) and a control without Cu for 20 days. Previous to laboratory acclimation, 8 fish were taken to define the initial population. At the end of the trials, 12 fish/sex/treatment were sampled for the determination of levels of copper in gills, metallothioneins (MTs) content, total lipids, fatty acids profiles and antioxidant enzymatic activity, as well as lipid peroxidation. Most of the antioxidant enzymatic defenses assayed were not activated and lipid peroxidation decreased significantly in fish exposed to any concentration of copper applied. This leads us to presume the existence of a protective mechanism against peroxidation other than the enzymatic antioxidant defense, which could be related to the observed increase of copper content in the gills.