Examination of pulse-labelled Newcastle disease virus (NDV)-infected chick embryo fibroblasts (CEF) by two-dimensional polyacrylamide gel electrophoresis revealed the presence of two-virus-coded non-structural polypeptides of mol. wt. 36K and 33K. Longer pulses and pulse-chase incubations revealed the production of an additional, glycosylated, non-structural polypeptide of mol. wt. 40K (gp40). Kinetic arguments suggest that 36K and 33 K are primary translation products but that gp40 is not. 36K was stable in chase incubations, but 33K was not. Partial digest peptide analysis showed that gp40 and an additional glycosylated polypeptide gp62, which is sometimes present (Chambers & Samson, 1980), are related to the HN polypeptides. Partial digest peptide analysis of the 36K polypeptide generated only a few peptides, which were not sufficient to conclude whether 36K was related to the major virus polypeptides, and since polypeptides 33K was metabolically unstable, insufficient radioactivity was incorporated for peptide studies. Extensive strain-dependent variation in the isoelectric points and mol. wt. of all the NDV polypeptides which are soluble in the isoelectric focusing gels, including 36K and 33K, is reported. This variation, and the insensitivity of the synthesis of 36K and 33K to actinomycin D, show that both non-structural polypeptides are virus-coded.