Anchorless fusion (F) proteins () of human respiratory syncytial virus (RSV) are seen by electron microscopy as unaggregated cones when the proteolytic cleavage at two furin sites required for membrane-fusion activity is incomplete, but aggregate into rosettes of lollipop-shaped spikes following cleavage. To show that this aggregation occurred by interactions of the fusion peptide, a deletion mutant of lacking the first half of the fusion peptide was generated. This mutant remained unaggregated even after completion of cleavage, supporting the notion that aggregation of involved the fusion peptide. As exposure of the fusion peptide is a key event that occurs after activation of F proteins, the uncleaved and cleaved forms of may represent the pre- and post-active forms of RSV F protein. In an analysis of the structural differences between the two forms, their thermostability before and after proteolytic cleavage was examined. In contrast to other viral proteins involved in membrane fusion (e.g. influenza haemagglutinin), the pre-active (uncleaved) and post-active (cleaved) forms of were equally resistant to heat denaturation, assessed by spectrofluorimetry, circular dichroism or antibody binding. These results are interpreted in terms of the proposed structural changes associated with the process of membrane fusion mediated by RSV F protein.