Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. Sphingosine 1-phosphate is a bioactive sphingolipid that regulates crucial physiological processes. We have recently reported that sphingosine 1-phosphate and sphingosine kinase are involved in a novel signaling pathway leading to acrosomal exocytosis (Suhaiman L et al., J Biol Chem 285:1630-16314, 2010). Acrosomal exocytosis in mammalian sperm is a regulated secretion with unusual characteristics. We therefore employed biochemical functional assays to assess the sphingolipid signaling in both permeabilized and nonpermeabilized sperm. The exocytosis of the acrosomal content is regulated by Ca(2+). During exocytosis, changes in [Ca(2+)]i occur induced by either Ca(2+)-influx or Ca(2+)-mobilization from intracellular stores. By using single cell [Ca(2+)] measurements, we detected intracellular Ca(2+) changes after sphingosine 1-phosphate treatment. Additionally, measuring sphingosine kinase activity, we determined that sphingosine 1-phosphate levels increase after an exocytotic stimulus.This chapter is designed to provide the user with sufficient background to analyze sphingosine 1--phosphate signal transduction pathways during acrosomal exocytosis in human sperm.