Ca2+ contributes to a myriad of important cellular processes in all organisms, including the apicomplexans, Plasmodium and Toxoplasma. Due to its varied and essential roles, free Ca2+ is tightly regulated by complex mechanisms. These mechanisms are therefore of interest as putative drug targets. One pathway in Ca2+ homeostatic control in apicomplexans uses a Ca2+/H+ exchanger (a member of the cation exchanger family, CAX). The P. falciparum CAX (PfCAX) has recently been characterised in asexual blood stage parasites. To determine the physiological importance of apicomplexan CAXs, tagging and knock-out strategies were undertaken in the genetically tractable T. gondii and P. berghei parasites. In addition, a yeast heterologous expression system was used to study the function of apicomplexan CAXs. Tagging of T. gondii and P. berghei CAXs (TgCAX and PbCAX) under control of their endogenous promoters could not demonstrate measureable expression of either CAX in tachyzoites and asexual blood stages, respectively. These results were consistent with the ability of parasites to tolerate knock-outs of the genes for TgCAX and PbCAX at these developmental stages. In contrast, PbCAX expression was detectable during sexual stages of development in female gametocytes/gametes, zygotes and ookinetes, where it was dispersed in membranous networks within the cytosol (with minimal mitochondrial localisation). Furthermore, genetically disrupted parasites failed to develop further from "round" form zygotes, suggesting that PbCAX is essential for ookinete development and differentiation. This impeded phenotype could be rescued by removal of extracellular Ca2+. Therefore, PbCAX provides a mechanism for free living parasites to multiply within the ionic microenvironment of the mosquito midgut. Ca2+ homeostasis mediated by PbCAX is critical and suggests plasmodial CAXs may be targeted in approaches designed to block parasite transmission. © 2013 Guttery et al.