Penetration of the oocyte by a spermatozoon is the first in the series of events resulting in the transition of the egg from a quiescent to a proliferative state. A critical regulatory role for intracellular calcium ([Ca2+](i)) ion activity has been demonstrated in all species studied so far. On the other hand, it has been demonstrated that the intracellular pH (pH(i)) changes, but only in a small number of species. This change also has been proposed as one of the most important events in egg activation. The present study was undertaken to monitor pH(i) in rat eggs during fertilization, using the membrane-permeable indicator BCECF-AM and fluorescence ratio imaging. Furthermore, we proposed to evaluate the relationship between pH(i) and [Ca2+](i) changes during egg activation. We found that the ovulated rat egg has a cytoplasmic pH significantly different from that of the follicular oocyte. Insemination with capacitated sperm resulted in a microscopically visible sperm attachment, yet no change in pH(i) was observed. Eggs double-loaded with fura-2-AM and BCECF-AM before insemination were used to measure [Ca2+](i) and pH(i) simultaneously. Eggs with a normal pattern of [Ca2+](i) transients (i.e., fertilized eggs) did not show any change in pH(i) at least for 30 min following sperm binding. Data for eggs fertilized in vivo were recorded at later times after sperm binding; these served to exclude the possibility of a transient change that occurs between sperm-egg interaction and the pronuclear stage. We conclude that the pH(i) of rat eggs does not change during fertilization and therefore that fertilization-induced [Ca2+](i) changes do not affect pH(i) in these eggs.