Fusion of sperm and egg plasma membranes is an early and essential event at fertilization but it is not known if it plays a part in the signal transduction mechanism that leads to the oscillations in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) that accompany mammalian egg activation. We have used two independent fluorescence methods and confocal microscopy to show that cytoplasmic continuity of egg and sperm precedes the onset of the first [Ca²⁺]_i increase in mouse eggs. The Ca²⁺ indicator dye Ca²⁺-green dextran was microinjected and its transfer from egg to sperm was monitored. We found that it occurred before, and without a requirement for, any detectable [Ca²⁺]_i increase in the egg. In separate experiments [Ca²⁺]_i changes were recorded in populations of eggs, using fura red, and the eggs fixed at various times after some of the eggs had shown a [Ca²⁺]_i transient. Fusion of the sperm and egg was then assessed by Hoechst dye transfer. All eggs that showed a [Ca²⁺]_i increase had a fused sperm but more than half of the eggs contained a sperm but had not undergone a [Ca²⁺]_i increase. These data indicate that sperm-egg fusion precedes [Ca²⁺]_i changes and we estimate that the elapsed time between sperm-egg fusion and the onset of the [Ca²⁺]_i oscillations is 1-3 minutes. Finally, sperm-egg fusion was prevented by using low pH medium which reversibly prevented [Ca²⁺]_i oscillations in eggs that had been inseminated. This was not due to disruption of signalling mechanisms, since [Ca²⁺]_i changes still occurred if low pH was applied after the onset of oscillations at fertilization. [Ca²⁺]_i changes also occurred in eggs in low pH in response to the muscarinic agonist carbachol. These data are consistent with the idea that the [Ca²⁺]_i signals that occur in mammalian eggs at fertilization are initated by events that are closely coupled to the fusion of the sperm and egg membranes.