Malaria parasite replicates within its own plasma membrane, positioned inside a parasitophorous vacuole that is located within the host human erythrocyte. Both the vacuolar and erythrocyte plasma membranes must be breached by the parasites to exit the host cell (see reviews by V Lew [
1,
2]. Experimental evidence supports a multistep process [
3‐
9] involving at least two Ca
2+-dependent enzymes: protein kinase PfCDPK5 [
5] and the host protease calpain [
4]. The emerging complexity of the egress mechanism suggests a high degree of spatial and temporal coordination. Cytosolic Ca
2+ is a universal secondary messenger for intracellular signalling in eukaryotic cells (for review see [
10]). Ca
2+ signalling in malaria parasites is well recognized but poorly understood; several signalling pathways have been described [
11‐
14]; for a recent review see [
15]. The total erythrocyte calcium content increases with infection [
16,
17]. However, it is harder to determine the free calcium concentration, [Ca
2+, in the various compartments of an infected erythrocyte at the schizont stage of the asexual cycle. Divided and tightly packed parasites occupying approximately half of the erythrocyte volume [
18], are situated within a series of concentric membrane spheroids; the individual parasites have additional levels of membrane compartments (organelles) internal to their own plasma membrane. Literature estimates of [Ca
2+ in different compartments range from low nM in the erythrocyte cytoplasm [
19] to ~ 40 μM in the parasitophorous vacuole – the space between the PV membrane and the parasite plasma membrane [
13]. Presumably, there are compartments within the parasite that maintain even higher Ca
2+ concentrations and can act as intracellular Ca
2+ stores [
11]. Thus, there is a complex set of potential gradients for Ca
2+ translocations between compartments with differing [Ca
2+. An influx of extracellular Ca
2+ into erythrocytes is required for parasite invasion [
20‐
22], however, the role Ca
2+ plays in the multistep egress programme is not clear [
21,
23]. The role of intracellular Ca
2+ is intensely studied in the egress programme of the related
Apicomplexan parasites,
Toxoplasma[
24‐
29]. However, sufficient differences in the molecular mechanisms of the egress pathway between this species and the malaria parasites are emerging [
28,
30]. This study reports the measurement of intracellular fluorescent probes for [Ca
2+ during the egress pathway of
Plasmodium falciparum from infected erythrocytes. Perturbation with calcium homeostasis reagents together with quantitative measures of egress that decouple egress from invasion allowed a determination of one source for the calcium fluxes that must occur for [Ca
2+ to change. Finally, to determine which of the observable stages of the parasite egress programme [
7] are sensitive to changes in [Ca
2+, the morphology of treated cells was analysed.