The biologically active form of the fusion glycoprotein F from Newcastle disease virus (NDV) comprises 2 polypeptides, F1 and F2 (derived from a precursor polypeptide F0 by a post translational cleavage event), which are covalently linked together (F1,2) by disulfide bonds. This feature was exploited in a 2-dimensional SDS[sodium dodecyl sulfate]-polyacrylamide gel electrophoretic analysis to orientate the position of the cleavage event within F0. Separation of proteins from NDV-infected CEF [chick embryo fibroblast] in the first dimension in the absence of reducing agent resolved F1,2 protein from all NDV-induced proteins other than F0. Reduction of the first dimension gel with 2-mercaptoethanol, followed by electrophoresis in the 2nd dimension, resolved F1 (55K [kilodalton]), F2 (12.5K) and F0 (64K) proteins. The only polypeptides other than F1 and F2 which fell below the diagonal, indicating the positions of the polypeptides from infected cells, were 2 minor glycoproteins designated HN1 (51.5K) and HN2 (27.5K), which took up positions vertically beneath the major hemagglutinin-neuraminidase glycoprotein HN (75K). Dual isotope labeling experiments with NDV-infected CEF, which had previously received a salt shock to effect synchronization of polypeptide initiation upon release of salt shock, revealed the following orientations within the parent molecules: NH2.sbd.F2.sbd.F1.sbd.COOH and NH2.sbd.HN1.sbd.HN2.sbd.COOH. The existence of intermolecular disulfide bonds, orientation and relative lengths of the 2 NDV HN fragments is analogous to the HA1 and HA2 proteins of influenza virus hemagglutinin.