Cytosolic Ca2+ ions play a central role as a universal intracellular messenger, controlling many aspects of physiology such as gene expression, electrical excitability, secretion and contraction. The rich diversity in information encoded by Ca2+ arises through the mechanisms by which Ca2+ signals are generated and transmitted to act over very different spatial and temporal scales. In turn, the spatio-temporal patterning and transmission of Ca2+ signals involve the inherent properties of the Ca2+ channels themselves, their spatial organization, and how these channels interact via diffusion of Ca2+ both with themselves and with effector proteins. This is exemplified by the inositol trisphosphate receptor/channel (IP3R), the opening of which requires binding of IP3 together with Ca2+ to cytosolic receptor sites (Foskett et al., 2007). Gating by Ca2+ is biphasic, creating both positive and negative feedback loops, whereby Ca2+ liberated through one channel may modulate its own opening and that of neighboring channels. Channel-channel interactions are delimited by the spatial arrangement of channels, and IP3Rs are organized into tight clusters on the ER membrane. This leads to a hierarchy of Ca2+ signals of differing magnitudes, kinetics and spatial extent (Callamaras et al., 1998): ‘Fundamental’ signals (Ca2+ blips) representing Ca2+ flux through individual channels; ‘elementary’ Ca2+ transients (Ca2+ puffs) generated by openings of multiple IP3R within a cluster, orchestrated by Ca2+-induced Ca2+ release (CICR); and propagating global waves of Ca2+ resulting from progressive recruitment of multiple clusters by successive cycles of Ca2+ diffusion and CICR. Electrophysiological patch-clamp recording – the gold standard for studying single channels under tightly controlled ionic and electrical conditions – is thus poorly suited to studies of the coordinated behavior of multiple, clustered Ca2+ channels in the intact cellular environment. Instead, our knowledge of Ca2+ signaling at the cellular and sub-cellular levels derives largely from optical imaging in intact cells employing fluorescent Ca2+ indicator dyes. Nevertheless, limitations of imaging technology have, until recently, precluded detailed study of single-channels and of channel-channel interactions within clusters which are too small to resolve by conventional light microscopy. This has now changed. Developments in optical imaging, particularly the application of total internal reflection fluorescence (TIRF) microscopy, enable the recording of single Ca2+ channel activity within live cells (Smith & Parker, 2009). Moreover, the wavelength of light no longer limits resolution: optical microscopy has become nanoscopy Patterson et al., 2010). We can localize Ca2+ channels at nanometer scales and study how their function is influenced by their spatial distribution. I will describe our use of these innovative techniques to take studies of Ca2+ signaling in cultured cell lines down to the truly single-molecule level, and discuss their application to studies in smooth muscle cells.