Seven differences in the polypeptide species of parental Syrian hamster embryo cells and cells of the highly tumorigenic derivative cell line BP6T were identified previously by employing the technique of two-dimensional polyacrylamide gel electrophoresis (Leavitt, J. and Moyzis, R. (1978) J. Biol. Chem. 253, 2497-2500). To determine which of these polypeptide changes are correlated with expression of the neoplastic state this work was extended to the comparative examination of nine established neoplastic cell lines which resulted from independent transformation events catalyzed by chemical carcinogen treatment, virus infection, or an unknown spontaneous event. Although no perfect correlation with a specific polypeptide change was found, two polypeptide changes, occurring independently or simultaneously, appear to be consistently associated with expression of neoplasticity. One polypeptide species, designated tau, having an isoelectric point of 4.6 and a molecular weight of 60 000 was lost or physically altered in all but one of these transformed cell lines; a second polypeptide species designated nu having an isoelectric point 5.5 and a molecular weight of 42 000 appeared in highly tumorigenic chemically transformed cell lines and in two virally transformed cell lines. A butyric acid supplement, used as a selective agent for butyric acid resistant cells, was employed to identify and isolate in a single step nascent neoplastic clonal lines transformed by ethylmethanesulfonate. These cell lines exhibited alterations either in tau or nu. The changes observed in tau are consistent with those expected to result from a somatic mutation event in the structural gene coding for tau; however, the alterations in tau could also be governed by a post-translational process. These findings suggest that alterations in expression of at least two major polypeptide species, tau and nu, are closely associated with primary steps in the neoplastic transformation process of Syrian hamster cells irrespective of the nature of the transforming agent.