One of the leading causes of male infertility is defective sperm function, a pathology that commonly arises from oxidative stress in the male germline. Oxidative stress is induced through reactive oxygen species (ROS), leading to lipid peroxidation, organelle degradation, DNA damage and apoptosis. Lipid peroxidation of the sperm plasma membrane results in the generation of cytotoxic aldehydes such as 4-hydroxynonenal (4HNE), which further elevate ROS production and oxidative stress. To investigate the specific mechanisms by which 4HNE is produced in developing germ cells, comparative proteomics was performed on isolated round spermatids exposed to 4HNE and their untreated counterparts. This study revealed a highly significant, 28-fold increase in the enzyme 15 arachidonate lipoxygenase (15-ALOX) following exposure of the male germ line to 4HNE. The 15-ALOX protein belongs to a family of non-heme iron containing enzymes implicated in protein signalling and lipid oxygenation. The latter of these roles has the potential to exacerbate ROS production and hence accentuate the level of oxidative stress experienced by the cell. Given this, we sought to characterise 15-ALOX in the developing germ cells of the mouse testis and in mature human and mice spermatozoa. The 15-ALOX enzyme was found to be highly expressed in pachytene spermatocytes and round spermatids and under conditions of oxidative stress, co-localisation of 15-ALOX and 4HNE was observed in the cell cytoplasm. Additionally, 15-ALOX was localised to the peri-acrosomal region of mature human and mouse spermatozoa, a domain that is critical for the mediation of sperm-oocyte interactions. These preliminary studies provide the impetus for further work focusing on the physiological role of 15-ALOX in the male germline. Specifically, we aim to explore the contribution of this enzyme to 4HNE production and to determine whether the inhibition of lipoxygenase function could afford protection for sperm cell function under conditions of oxidative stress.