Live-cell imaging chambers are used in a wide range of cell biology research. Recently, chambers capable of taking high-resolution and time-lapse images of live cells have been developed and become commercially available. However, because most of these chambers are designed to maintain a thermally stable environment for the cells under study, it is usually very difficult to use them to study temperature-dependent cellular events. Here we report the development of a chamber that is able to be used for the continuous monitoring of live neurons under most commercially available upright epifluorescence and confocal microscopes and in which the temperature and composition of the medium surrounding the neurons can be changed rapidly and reversibly. This live-cell observation chamber has been used successfully with cultured rat hippocampal neurons to study temperature-dependent changes in the dynamics of the microtubule cytoskeleton using fluorescence recovery after photobleaching (FRAP) together with the localization of alpha-tubulin in the dendritic spines. The success of these observations demonstrates the usefulness and applicability of the live-cell observation chamber described here to a wide range of cell biology experiments.