In both the mouse and the human, it is a point of controversy whether glucose is necessary for in vitro fertilization. Some of this controversy has resulted from a failure to distinguish between requirements for glucose during sperm capacitation versus requirements during the multistage process of fertilization. Using the mouse as a model, we performed a series of experiments designed to identify specific processes that might require glucose. We observed a positive correlation between increasing glucose concentrations during capacitation and fertilization, and increasing fertilization of zona pellucida (ZP)-intact eggs. These data supported a requirement for glucose in the fertilization medium even when sperm were first capacitated in the presence of 5.5 mM glucose. This glucose requirement was observed for both ZP-intact and ZP-free eggs. During ZP-free in vitro fertilization, some binding and fusion between the plasma membrane of the sperm and egg occurred in the absence of glucose and at concentrations less than 1 mM, suggesting that this substrate is not absolutely required. However, glucose concentrations of 1 mM or higher greatly facilitated both binding and fusion under these conditions. These subtle distinctions suggest that during ZP-free in vitro fertilization, 1 mM glucose represents a threshold level that facilitates binding and fusion. Taken as a whole, the data suggest requirements for glucose during both capacitation and fertilization under normal physiologic conditions.