Proteomics represents a powerful tool for the analysis of mammalian spermatozoa, since these terminally differentiated cells are transcriptionally inactive and exhibit a limited dynamic range of protein expression. Here we report the identification of 5123 peptides, leading to 829 unambiguous and 2215 redundant gene products found to be present within rat spermatozoa derived from the cauda epididymis. Bioinformatics demonstrated that 60 proteins appeared to be specifically expressed in the genitourinary tract, including pyruvate dehydrogenase 1, ropporin, testis-specific serine kinase 4, testis-specific transporter, and retinol dehydrogenase 14. We also identified eight members of the ADAM family, seven of which have previously been detected in spermatozoa (ADAM2, -3, -4, -5, -6, -7, and -30) while ADAM34 has been identified in the sperm proteome for the first time. Approximately 21 gene products were found to possess isomerase activity including peptidylprolyl cis/trans isomerases that are known to be involved in germ cell differentiation and protein disulfide isomerases that have been implicated in sperm–oocyte fusion. Furthermore, 51 gene products clustered into ion-transporter activity. This inventory of gene products, the first ever 2-D LC-MS/MS analysis of rat spermatozoa, will be invaluable in directing future research into the molecular mechanisms that drive these highly specialized cells.