The products of the ras genc family are related proteins at a molecular weight of 2 I kDa, designated p2 I . In the present study we used two-dimcn~ional gcl electrophoresis to compare p2 1 proteins from fivc different normal and malignnnt cell lines. Using a known protein (%labeled translation initiation factor lclF-4D]) as a standard internal marker for isoelectric point (pl), we show that p?l proteins froin various cells differ only slightly in tiiolecular weight (2 1-24 hDa) but express a wide variety in charge (pl 4.8 to 7) that could only be detected by the use of two-dinicnsional gel electrophoresis. p21 in NIHi3T3 cells was expressed as a single protein, which migrated at 21 kDa and pl 5.1. This pcptidc. which is probably the product of the normal cellular ras gene. was also detected in normal human lymphocytes. The synthesis of this peptide was not elevated in the transformed cells. However, transformation of NIHi3T3 fibroblasts and of human leukocytes was found to be associated with expression of qualitatively different forms of p2 1 peptides. Four additional p2 1-associated peptides of identical molecular weight (23 kDa), but multiple charge forms, were detected selectively in Kirsten niurine sarconia virus-transformed NIHi3T3 cells. Transformation of cells with Harvey murine sarconia virus was found to be associated with prominent expression of two major pairs of p21-associated proteins. one at 21 kDa (pl. 5.2 and 5.3) and the other at 23 kDa (pl, 5.1 and 5.2). In HL-60 leukemic cells there was an additional. more acidic form (pl 5.0) of p21. which appeared to be absent or reduced in normal human lymphocytes. These results indicate that p21 from viral origin or cellular origin might be expressed in the cells in multiple charge forms. The capability to distinguish multiple forms of p2 1 and slight charge niodifications associated with malignancy should call for the use of 2-D gel electrophoresis as an important tool in future studies involving p2l proteins.