Chloroplasts require a fine-tuned control of their internal Ca2+ concentration, which is crucial for many aspects of photosynthesis and for other chloroplast-localized processes. Increasing evidence suggests that calcium regulation within chloroplasts also may influence Ca2+ signaling pathways in the cytosol. To investigate the involvement of thylakoids in Ca2+ homeostasis and in the modulation of chloroplast Ca2+ signals in vivo, we targeted the bioluminescent Ca2+ reporter aequorin as a YFP fusion to the lumen and the stromal surface of thylakoids in Arabidopsis (Arabidopsis thaliana). Thylakoid localization of aequorin-based probes in stably transformed lines was confirmed by confocal microscopy, immunogold labeling, and biochemical analyses. In resting conditions in the dark, free Ca2+ levels in the thylakoid lumen were maintained at about 0.5 μm, which was a 3- to 5-fold higher concentration than in the stroma. Monitoring of chloroplast Ca2+ dynamics in different intrachloroplast subcompartments (stroma, thylakoid membrane, and thylakoid lumen) revealed the occurrence of stimulus-specific Ca2+ signals, characterized by unique kinetic parameters. Oxidative and salt stresses initiated pronounced free Ca2+ changes in the thylakoid lumen. Localized Ca2+ increases also were observed on the thylakoid membrane surface, mirroring transient Ca2+ changes observed for the bulk stroma, but with specific Ca2+ dynamics. Moreover, evidence was obtained for dark-stimulated intrathylakoid Ca2+ changes, suggesting a new scenario for light-to-dark-induced Ca2+ fluxes inside chloroplasts. Hence, thylakoid-targeted aequorin reporters can provide new insights into chloroplast Ca2+ storage and signal transduction. These probes represent novel tools with which to investigate the role of thylakoids in Ca2+ signaling networks within chloroplasts and plant cells.