The recent identification of RNA as a component of mature spermatozoa necessitated the development of a reliable isolation protocol capable of yielding a high-quality substrate. In addition to the inherent difficulties associated with isolating RNA, the procedure as applied to sperm must overcome the resilient nature and reduced RNA content found within this cell type. Further, the protocol must be suited to the clinical setting. A reliable RNA isolation procedure optimized for this unique cell type is described. Ejaculate is collected, contaminating somatic cells lysed then spermatozoal RNA released by homogenization in a chaotrope. RNA is then purified from the homogenate by chromatography using a commercially available resin. The quality of isolated samples is assessed by PCR and RT-PCR. Once purity is established samples are suitable for numerous applications including amplification and probe synthesis. The reliable and consistent isolation of high-quality RNA from mature spermatozoa will aid in the development of new tools for the clinical assessment of male-factor fertility.