Using transgenic Nicotiana plumbaginifolia seedlings in which the calcium reporter aequorin is targeted to the chloroplast stroma, we found that darkness stimulates a considerable flux of Ca²⁺ into the stroma. This Ca²⁺ flux did not occur immediately after the light-to-dark transition but began 5 min after lights off and increased to a peak at 20 to 30 min after the onset of darkness. Imaging of aequorin emission confirmed that the dark-stimulated luminescence emanated from chloroplast-containing tissues of the seedling. The magnitude of the Ca²⁺ flux was proportional to the duration of light exposure (24 to 120 h) before lights off; the longer the duration of light exposure, the larger the dark-stimulated Ca²⁺ flux. On the other hand, the magnitude of the dark-stimulated Ca²⁺ flux did not appear to vary as a function of circadian time. When seedlings were maintained on a 24-h light/dark cycle, there was a stromal Ca²⁺ burst after lights off every day. Moreover, the waveform of the Ca ²⁺ spike was different during long-day versus short-day light/dark cycles. The dark-stimulated Ca²⁺ flux into the chloroplastidic stroma appeared to affect transient changes in cytosolic Ca²⁺ levels. DCMU, an inhibitor of photosynthetic electron transport, caused a significant increase in stromal Ca ²⁺ levels in the light but did not affect the magnitude of the dark-stimulated Ca²⁺ flux. This robust Ca²⁺ flux likely plays regulatory roles in the sensing of both light/dark transitions and photoperiod.