The adult population of Leydig cells acts to secrete testosterone which is essential for reproductive health and fertility in the adult male. Other physiological functions of these cells are uncertain, however, and to address this issue a cell-ablation model has been used to identify Leydig cell-specific mRNA transcripts. Ethane dimethane sulphonate (EDS) was synthesised by a novel process and was used to ablate Leydig cells in adult male rats previously treated with butane dimethane sulphonate (busulphan) to delete the germ cell population. Levels of mRNA transcripts were measured in the testis using microarrays 1, 3, 5, 8 and 12 days after EDS injection. During this period there was a significant change in the levels of 2,211 different transcripts with a marked decline in the levels of canonical Leydig cell transcripts such as Cyp11a1, Cyp17a1 and Insl3. A total of 95 transcripts showed a similar decline in expression after EDS suggesting that they have a Leydig cell-specific origin. Analysis of selected transcripts confirmed that they were expressed specifically in Leydig cells and showed that most had a late onset of expression during adult Leydig cell development. Apart from transcripts encoding components of the steroidogenic apparatus, the most common predicted function of translated proteins was endogenous and xenotoxicant metabolism. In addition a number of transcripts encode acute phase proteins involved in reduction of oxidative stress. Results show that, in addition to androgen secretion, Leydig cells may have a critical role to play in protecting the testis from damage caused by toxicants or stress.