Changes in intracellular free calcium ion concentration ([Ca(2+)](i)) have been visualized over more than two decades using fluorescent dyes and optical microscopy. So far, however, most imaging studies have been performed on isolated cells or brain tissue. Here, we review approaches to measure cellular [Ca(2+)](i) changes in vivo, i.e. within the intact brain of a living animal. In particular we describe the application of two-photon microscopy to the mammalian central nervous system, which has recently enabled studies of Ca(2+) dynamics in individual dendrites in anaesthetized rats. New developments in microscopy and labeling techniques are creating further opportunities to study Ca(2+) dynamics in vivo and are likely to make measurements of spatio-temporal [Ca(2+)](i) distributions feasible even in awake, behaving mammals.