The relationship between the length of the connecting peptide in a paramyxovirus F0 protein and cleavage of F0 into the F1 and F2 subunits has been examined by constructing a series of mutant F proteins via site-directed mutagenesis of a cDNA clone encoding the simian virus 5 F protein. The mutant F proteins had one to five arginine residues deleted from the connecting peptide. The minimum number of arginine residues required for cleavage-activation of the simian virus 5 F0 protein by host cell proteases was found to be four. F proteins with two or three arginine residues in the connecting peptide were not cleaved by host cell proteases but could be cleaved by exogenously added trypsin. The mutant F protein possessing a connecting peptide consisting of one arginine residue was not cleaved by trypsin. The altered F proteins were all transported to the infected-cell plasma membrane as shown by cell surface immunofluorescence or cell surface trypsinization. However, the only mutant F protein found to be biologically active as detected by syncytium formation was the F protein which has four arginine residues at the cleavage site. The results presented here suggest that in the paramyxovirus F protein the number of basic amino acid residues in the connecting peptide is important for cleavage of the precursor protein by host cell proteases but is not the only structural feature involved. In addition, the data indicate that cleavage of F0 into F1 and F2 does not necessarily result in biological activity and that the connecting peptide may affect the local conformation of the F polypeptide.