Signalling via G-protein-coupled receptors occurs in a temporally and spatially organized manner. However, conventional techniques offer very little resolution of this spatial and temporal patterning. In order to permit a better analysis of these processes, we have developed a series of fluorescent methods to record and image various steps in the sequence of signalling via G-protein-coupled receptors: ligand binding, receptor activation via their conformational change, receptor/G-protein interaction, G-protein activation (via subunit rearrangement), ion channel activation (by patch clamp analysis), and the generation of the second messenger cAMP. Our data show that ligand binding is rate limiting at low ligand concentrations, and that it may occur as a biphasic process. At higher ligand concentrations, most receptors activate within about 50 ms, and receptor/G-protein- interaction occurs with the same time course, suggesting virtually instantaneous interaction of activated receptors with G-proteins. In contrast, activation of various types of G-proteins is about ten-fold slower, with time constants in the range of 500 ms. G-protein activation appears to by tightly linked to effector activation such as monitored by opening of the GIRK K-channel. Increases in the concentrations of the second messenger cAMP occur over seconds to minutes. In most cells, cAMP appears to diffuse freely throughout the cytosol, enabling ubiquitous responses. However, in cardiomyocytes cAMP does not seem to diffuse freely, and cAMP signals caused by stimulation of beta2-adrenergic recepors appear to stay localized, whereas those initiated by beta1-receptor stimulation are more generalized. Simultaneous recording of different second messengers indicate complex interactions between the levels of cAMP, cGMP and calcium that can be mediated both by effects on the generation and on the degradation of these messengers. Taken together, our studies show a differentiated pattern of signal propagation along signalling chains, and and a great complexity of the spatial and temporal characteristics of the signals transduced via these pathways. The physiological relevance of such distinct patterns remains to be elucidated, but it is clear that they permit a much better encoding of biological messages than previously anticipated.