Fertilization is an exceptionally specific cell recognition event that represents the culmination of a complex sequence of morphological and functional maturational events. In the case of the male gamete, this process is initiated by the commitment of spermatogonial stem cells to differentiate, sequentially forming spermatogonia, spermatocytes and eventually spermatozoa that are released into the lumen of the seminiferous tubules [1]. In addition to meiotic divisions, this process encompasses extensive cytoplasmic, organelle and nuclear remodeling events, thus establishing the unique and highly polarized architecture of the mature spermatozoon. A key aspect of this phase of development is the modification and repositioning of the Golgi apparatus to form a highly specialized secretory organelle, known as the acrosome, overlying the anterior aspect of the sperm head. Upon release from the testes the functionally immature spermatozoa enter the epididymis where they are progressively remodeled and acquire both motility and the potential to fertilize an oocyte [2]. This potential is eventually realized after passage through the female reproductive tract whereupon the ejaculated cells complete a suite of biochemical and biophysical changes known as capacitation [3]. These successive phases of functional maturation culminate in the acquired ability to release the acrosomal contents, during an event known as the acrosome reaction. This unique exocytotic event facilitates sperm passage through the outer vestments of the oocyte and is essential for successful in vivo fertilization in all mammalian species, including the human [4]. Consequently, failure of acrosomal exocytosis represents a common etiology in defective spermatozoa of male infertility patients that have failed in vitro fertilization (IVF) in a clinical setting; accounting for as much as 29 percent of unexplained male infertility cases [5, 6]. Much of our current mechanistic understanding of the acrosome reaction is grounded in the ability to stimulate this process in vitro using simple chemically defined media and the application of pharmacological interventions, and/or transgenic mouse models. Here, we discuss the biological significance of the acrosome reaction and the application of histochemical techniques that have been developed to study the progression and completion of this critical physiological event.