Transient spontaneous increases in the intracellular Ca2+ concentration have been frequently observed in astrocytes in cell culture and in acutely isolated slices from several brain regions. Recent in vivo experiments, however, reported only a low frequency of spontaneous Ca2+ events in astrocytes. Since the ex vivo experiments were usually performed at temperatures lower than physiological body temperature, we addressed the question whether temperature could influence the spontaneous Ca2+ activity in astrocytes. Indeed, comparing the frequency and spike width of spontaneous Ca2+ transients in astrocytes at temperatures between 20 and 37 degrees C in culture as well as in acute cortical slices from mouse brain, revealed that spontaneous Ca2+ responses occurred frequently at low temperature and became less frequent at higher temperature. Moreover, the single Ca2+ events had a longer duration at low temperature. We found that nitric oxide (NO) mimicked the increase in spontaneous Ca2+ activity and that an NO-synthase inhibitor attenuated the effect of lowering the temperature. Thus, temperature and NO are major determinants of spontaneous astrocytic Ca2+ signalling.