Membrane fusion constitutes an essential, intermediate step in numerous cell biological processes, occurring for example during endocytosis, membrane recycling and exocytosis. Also less desirable events such as the infection of cells by animal viruses are mediated by membrane fusion during which the viral envelope merges with a cellular membrane, causing the expulsion of the viral nucleocapsid into the cytoplasm of the cell as an initial step in virus replication. Much of our current knowledge concerning the mechanism of membrane fusion has been derived from studies using simple artificial membranes, such as liposomes or phospholipid vesicles, as model systems. A most essential feature of these studies has been the development of membrane fusion assays that register in a sensitive and continuous fashion the mixing of membranes or the aqueous volumes initially enclosed by these membranes. Not only do these assays allow one to readily detect and quantify fusion, but they also provide the possibility to relate the kinetics of fusion to the rate by which certain molecular changes in membranes take place. Obviously, this insight is of relevance for understanding the mechanism of membrane fusion. The principles and applications of some representative assays that rely on the use of fluorescence spectroscopy will be discussed. Assays that monitor membrane mixing are commonly based on the detection of changes in resonance energy transfer efficiency or the relief of fluorescence self-quenching of appropriate fluorescent lipid analogs. Contents mixing assays rely on either the formation of a (aqueous-soluble) fluorescent complex or quenching of a fluorophore, encapsulated in one vesicle population, by a suitable quencher, entrapped in a second population.(ABSTRACT TRUNCATED AT 250 WORDS)