Analysis of the respiratory syncytial virus (RSV) fusion (F) protein in RSV-infected Vero cells showed the presence of a single F1 subunit and at least two different forms of the F2 subunit, designated F2a (21 kDa) and F2b (16 kDa), which were collectively referred to as [F2](a/b). Enzymatic deglycosylation of [F2](a/b) produced a single 10 kDa product suggesting that [F2](a/b) arises from differences in the glycosylation pattern of F2a and F2b. The detection of [F2](a/b) was dependent upon the post-translational cleavage of the F protein by furin, since its appearance was prevented in RSV-infected Vero cells treated with the furin inhibitor dec-RVKR-cmk. Analysis by protein cross-linking revealed that the F1 subunit interacted with [F2](a/b), via disulphide bonding, to produce equivalent F protein trimers, which were expressed on the surface of infected cells. Collectively, these data show that multiple F protein species are expressed in RSV-infected cells.