Bovine respiratory syncytial (BRS) virus is an important cause of serious respiratory illness in calves. The disease caused in calves is similar to that caused by human respiratory syncytial (HRS) virus in children. The two viruses, however, have distinct host ranges and the attachment glycoproteins, G, have no antigenic cross-reactivity. The fusion glycoproteins, F, of the HRS and BRS viruses, however, have some antigenic cross-reactivity. To further compare the BRS virus and HRS virus fusion proteins, we determined the nucleotide sequence of cDNA clones to the BRS virus F protein mRNA, deduced the amino acid sequence, and compared these sequences with the HRS virus F protein sequences. The BRS virus F mRNA was 1899 nucleotides in length and had a single major open reading frame which could code for a polypeptide of 574 amino acids with an estimated molecular weight of 63.8 kDa. Structural features predicted from the amino acid sequence included an NH2-terminal signal sequence (residues 1-26), a site for proteolytic cleavage (residues 131-136) to generate the disulfide-linked F1 and F2 subunits, and a hydrophobic transmembrane anchor sequence (residues 522-549). The nucleic acid identity between the BRS virus and the HRS virus F mRNA sequences was 71.5%. The predicted BRS virus F protein shared 80.5% overall amino acid identity with the HRS virus F protein with 89% identity in the F1 polypeptide but only 68% identity in the F2 polypeptide. The position and number of the cysteine residues in the F1 and F2 polypeptides were conserved among all F proteins. However, BRS virus F protein had only three potential N-linked carbohydrate acceptor sites in comparison to four or five for the HRS viruses. A difference in the extent of glycosylation between the BRS and HRS virus F2 polypeptides was shown to be responsible for differences observed in the electrophoretic mobility of these proteins. A cDNA containing the complete open reading frame of the BRS virus F mRNA was inserted into the thymidine kinase gene of vaccinia virus and following homologous recombination, a recombinant virus containing the BRS virus F gene was isolated. The BRS virus F protein was expressed in recombinant virus infected cells as demonstrated by immunoprecipitation and was transported to and expressed on the surface of infected cells as shown by indirect immunofluorescence.