Ca²⁺ uptake and release from endoplasmic reticulum (ER) and mitochondrial Ca²⁺ stores play important physiological and pathological roles, and these processes are shaped by interactions that depend on the structural intimacy between these organelles. Here, we investigate the morphological and functional relationships between mitochondria, ER and the sites of intracellular Ca²⁺ release in Xenopus laevis oocytes by combining confocal imaging of local Ca²⁺ release events ('Ca²⁺ puffs')with mitochondrial localization visualized using vital dyes and subcellularly targeted fluorescent proteins. Mitochondria and ER are both localized in cortical bands ~6µm wide, with the mitochondria arranged as densely packed 'islands'interconnected by discrete strands. The ER is concentrated more superficially than mitochondria, and the mean separation between Ca²⁺ puff sites and mitochondria is ~ 2.3µm. However, a subpopulation of Ca²⁺ puff sites is intimately associated with mitochondria (~ 28% within < 600 nm) - a greater number than expected if Ca²⁺ puff sites were randomly distributed. Ca²⁺ release sites occurring in close proximity to mitochondria exhibit lower Ca²⁺ puff activity than Ca²⁺ puff sites in regions with lower mitochondrial density. Furthermore, Ca²⁺ puff sites in close association with mitochondria rarely serve as the sites for Ca²⁺ wave initiation. We conclude that mitochondria play important roles in regulating local ER excitability, Ca²⁺ wave initiation and thereby the spatial patterning of cellular Ca²⁺ signals.