A series of prolonged intracellular Ca²⁺ oscillations are responsible for activating the development of mammalian eggs (Stricker, 1999). These Ca²⁺ oscillations have been shown to cause increases in mitochondrial NADH and FADH2 (Dumollard et al. 2004), but the effects upon cytosolic ATP levels are unclear. Here, we have monitored both intracellular Ca²⁺, using Oregon Green BAPTA dextran fluorescence, and ATP concentrations using luminescence from firefly luciferase. These indicators were microinjected into mouse eggs and both fluorescent and luminescent images were collected by using a cooled intensified CCD camera based system that allowed for intermittent fluorescent excitation. Data are presented as the mean and SD. During in vitro fertilization of mouse eggs we found that Ca²⁺ oscillations were associated with a two-phase rise in luciferase luminescence (131±9.8% for the 1st phase and 161±18.8% for the 2nd phase, n=34). Both increases were significantly different from luminescence levels recorded over a similar time period in unfertilized eggs (unpaired t test, P<0.005). This suggests a twofold increase in ATP levels at fertilization from 1.88mM to a peak of 3.02mM. The initial ATP increase started during the first Ca²⁺ transient at fertilization and peaked 30 min after oscillations began. The secondary increase in ATP typically occurred 1 h after the start of Ca²⁺ oscillations and was inhibited by nocodazole which blocks meiotic resumption (n=12). The ATP levels slowly returned to prefertilization levels after the cessation of Ca²⁺ oscillations. The ATP increase at fertilization was dependent upon Ca²⁺ because fertilization of eggs that had been injected with the Ca²⁺ chelator BAPTA (n=9) blocked both Ca²⁺ oscillations and the rise in luminescence. Stopping ongoing Ca²⁺ oscillations at fertilization by chelation of extracellular Ca²⁺ caused a decline in ATP back to prefertilization levels (n=11). Furthermore, ATP increases could be stimulated by stimuli that cause Ca²⁺ oscillations such as addition of extracellular carbachol, or intracellular injection of PLCzeta cRNA. Repetitive Ca²⁺ increases could also be induced by pulses of UV light delivered to eggs that had been injected with caged InsP3 (Jones & Nixon, 2000). Such repetitive InsP3-induced Ca²⁺ increases stimulated at intervals of 10 min or 5 min resulted in a slow rise in luciferase luminescence (114±7.9%, n=6, and 119±8.7%, n=10, respectively). However, Ca²⁺ transients induced by InsP3 uncaging at intervals of 2.5 min resulted in a decrease in luciferase luminescence (-14.5±5.9%, n=8). These data suggest that Ca²⁺ oscillations at fertilization cause a net rise in ATP that starts with the first Ca²⁺ transient.