The fusion of lipid bilayers can be visualized under the fluorescence microscope, but the process is very fast and requires special techniques for its study. It is reported here that vesicle fusion is susceptible to analysis by microspectrofluorometry and that for the first time, the entire fusion process has been captured. In the case of giant (>10- micro m diameter) bilayer vesicles having a high density of opposite charge, fusion proceeds through stages of adhesion, flattening, hemifusion, elimination of the intervening septum, and uptake of excess membrane to generate a spherical product very rapidly. These investigations became possible with a fluorescence microscope that was modified for recording of images simultaneously with the collection of fluorescence emission spectra from many (>100) positions along the fusion axis. Positively-charged vesicles, composed of O-ethylphosphatidylcholine and dioleoylphosphatidylcholine, were labeled with a carbocyanine fluorophore. Negatively-charged vesicles, composed of dioleoylphosphatidylglycerol and dioleoylphosphatidylcholine, were labeled with a rhodamine fluorophore that is a resonance energy transfer acceptor from the carbocyanine fluorophore. An electrophoretic chamber allowed selection of pairs of vesicles to be brought into contact and examined. Spectral changes along the axis of fusion were captured at high speed (a few ms/frame) by operating a sensitive digital camera in the virtual-chip mode, a software/hardware procedure that permits rapid readout of selected regions of interest and by pixel binning along the spectral direction. Simultaneously, color images were collected at video rates (30 frame/s). Comparison of the spectra and images revealed that vesicle fusion typically passes through a hemifusion stage and that the time from vesicle contact to fusion is <10 ms. Fluorescence spectra are well suited to rapid collection in the virtual-chip mode because spectra (in contrast to images) are accurately characterized with a relatively small number of points and interfering signals can be removed by judicious choice of barrier filters. The system should be especially well-suited to phenomena exhibiting rapid fluorescence change along an axis; under optimal conditions, it is possible to obtain sets of spectra (wavelength range of approximately 150 nm) at >100 positions along a line at rates >1000 frames/s with a spectral resolution of approximately 10 nm and spatial resolution at the limit of the light microscope ( approximately 0.2 micro m).