Abstract: N-acetyl-serotonin, the immediate precursor of melatonin in the tryptophan metabolic pathway in the pineal gland, has been reported to be an antioxidant. The aim of this study was to test the in vitro protective effect of N-acetyl-serotonin on the ascorbate-Fe++ induced lipid peroxidation of polyunsaturated fatty acids (PUFAs) located in testis microsomes and mitochondria. We assayed increasing concentrations (0–10 mm) of N-acetyl-serotonin in testis microsomes and (0–1 mm) of N-acetyl-serotonin in testis mitochondria. Control experiments were performed by incubating microsomal and mitochondrial membranes with N-acetyl-serotonin in the absence of lipid peroxidation-inducing drugs. Special attention was paid to the changes produced on the highly PUFAs C20:4 n6 and C22:5 n6. The light emission (chemiluminescence) used as a marker of lipid peroxidation was similar in both organelles when the control and peroxidized groups were compared. N-acetyl-serotonin reduced lipid peroxidation in testicular microsomes or mitochondria for both C20:4 n6 and C22:5 n6. Both long chain PUFAs were protected when N-acetyl-serotonin was incorporated either into microsomes or mitochondria. The N-acetyl-serotonin concentration required to inhibit by approximately 70% lipid peroxidation process was 10 mm in microsomes and between 0.50 and 1 mm in mitochondria. IC 50 values calculated from the inhibition curve of N-acetyl-serotonin on the chemiluminescence rates were higher in microsomes (4.50 mm) than in mitochondria (0.25 mm). In these experimental conditions, N-acetyl-serotonin was about 18 times more potent in testicular mitochondria in inhibiting the oxidative processes than it was in testicular microsomes. These results suggest that the protective role of N-acetyl-serotonin in preserving the long PUFAs may be related to its ability to reduce lipid peroxidation.