This paper presents a kinetic analysis of low-pH-induced fusion of Semliki Forest virus (SFV) with cholesterol-containing unilamellar lipid vesicles (liposomes), consisting otherwise of phosphatidylcholine, phosphatidylethanolamine and sphingomyelin. Fusion is monitored continuously with a lipid mixing assay, involving virus bio-synthetically labeled with the fluorophore pyrene. At pH 5.55, 37 degrees C, SFV-liposome fusion occurs on the time scale of seconds. Extensive fusion (up to 60% of the virus) requires an excess of liposomes, while a low-pH preincubation of the virus alone results in inactivation of its fusion capacity. The onset of fusion after acidification of virus-liposome mixtures is preceded by a pH- and temperature-dependent lag phase. Early in this lag phase, a conformational change in the E2E1 spike glycoprotein occurs, involving formation of a trypsin-resistant E1 homotrimer, exposing a conformation-specific epitope (E1"). These changes are followed by a rapid, cholesterol-dependent binding of the virus to the liposomes (as assessed by sucrose density gradient analysis), subsequent fusion starting only after an additional delay. This sequence of events strongly suggests that the E1 homotrimeric structure represents the fusion-active conformation of the SFV spike, the actual fusion complex possibly involving a higher order oligomer of E1 trimers.