The respiratory syncytial virus (RSV) fusion protein F is considered an attractive vaccine candidate especially in its prefusion conformation. We studied whether recombinant soluble RSV F proteins could be stabilized in a prefusion-like conformation by mutation of heptad repeat B (HRB). The results show that soluble， trimeric， non-cleaved RSV F protein， produced by expression of the furin cleavage site-mutated F ectodomain extended with a GCN4 trimerization sequence， is efficiently recognized by pre- as well as postfusion-specific antibodies. In contrast， a similar F protein completely lacking HRB displayed high reactivity with prefusion-specific antibodies recognizing antigenic site Ø， but did not expose postfusion-specific antigenic site I， in agreement with this protein maintaining a prefusion-like conformation. These features were dependent on the presence of the GCN4 trimerization domain. Absence of cleavage also contributed to binding of prefusion-specific antibodies. Similar antibody reactivity profiles were observed when the prefusion form of F was stabilized by the introduction of cysteine pairs in HRB. To study whether the inability to form the 6HB was responsible for the prefusion-like antibody reactivity profile， alanine mutations were introduced in HRB. Although introduction of alanine residues in HRB inhibited the formation of the 6HB， the exposure of postfusion-specific antigenic site I was not prevented. In conclusion， proteins that are not able to form the 6HB， due to mutation of HRB， may still display postfusion-specific antigenic site I. Replacement of HRB by the GCN4 trimerization domain in a non-cleaved soluble F protein resulted， however， in a protein with prefusion-like characteristics， suggesting that this HRB-lacking protein may represent a potential prefusion F protein subunit vaccine candidate.