Calcium imaging uses optical imaging techniques to measure the concentration of free calcium [Ca(2+)] in live cells. It is a highly informative technique in neurobiology because Ca(2+) is involved in many neuronal signaling pathways and serves as the trigger for neurotransmitter release. The technique relies on loading Ca(2+) indicators into cells, measuring the quantity and/or wavelength of the photons emitted by the Ca(2+) indicator, and interpreting these data in terms of [Ca(2+)]. There are several possible methods for loading synthetic Ca(2+) indicators into subcellular compartments, for example, topical application of membrane-permeant Ca(2+) indicators, forward-filling of dextran conjugates, and direct injection. These techniques are applicable to calcium imaging at the Drosophila larval neuromuscular junction (NMJ), and are also readily adaptable to Drosophila embryo and adult preparations.