Aromatic amino acids, which are present at high levels in egg yolk-containing cryodiluents, may be converted to damaging reactive oxygen species (ROS) by L-amino acid oxidase (LAAO) activity. The LAAO system is known to be present in bovine sperm, but it has not been reported in equine sperm. The objectives of this study were to characterize the LAAO activity in stallion sperm and evaluate the influence of aromatic amino acids on ROS production and sperm function. Using a luminol-peroxidase assay to measure H₂O₂ production, the responses of viable and non-viable spermatozoa to all the naturally occurring amino acids were tested. L-phenylalanine elicited the greatest responses in both viable and non-viable sperm (P<0.05). The only other significant responses were obtained with L-tryptophan and L-arginine in non-viable sperm. Immuno-cytochemical analysis showed that the LAAO activity was located in the sperm head, mainly in the acrosomal and post-acrosomal regions. Analyses of ROS generation in subcellular sperm fractions also found the majority of the LAAO activity to be located in the sperm head. Next, an examination of the cryomedia composition showed that aromatic amino acids were present at concentrations sufficient to activate the LAAO system and generate ROS. When viable and non-viable sperm were together exposed to aromatic amino acids at the levels found in cryomedia, total and progressive motility were both completely abolished (total: 28% v 0%; progressive: 14% v 0%), and lipid peroxidation, as measured by the percentage of 4-HNE positive cells, increased significantly (7% v 31%). In the final experiment, the addition of phenylglycine methyl ester derivatives, which are LAAO inhibitors, reduced the L-phenylalanine-induced production of H₂O₂, in both viable and non-viable sperm, in a dose-dependent manner (P<0.05). These results demonstrate that stallion sperm possess LAAO activity, and suggest that the aromatic amino acids present in commonly used equine cryomedia exert a negative effect on stallion sperm function.