Two-dimensional gel electrophoresis was used to detect [35S]methionine-labeled newly synthesized proteins from corticosteroid-sensitive and corticosteroid-resistant P1798 mouse lymphosarcoma cells. When the protein patterns from corticosteroid-sensitive cells are compared with those from highly resistant cells, they are very similar except for seven changes. One of the most obvious changes is the appearance of a new protein with a molecular weight of about 36,000 in highly resistant cells. There are also four other proteins that increase in the resistant state, as well as two that decrease. The control experiments indicate that protein differences are present to approximately the same degree when the sensitive tumor is carried in adrenalectomized animals, suggesting that the protein differences are not induced by endogenous glucocorticoids. Other experiments show that the protein differences are neither components of the small amounts of fibrous tissue that are associated with the cells nor components of blood. These results suggest that resistance of the tumor cells to glucocorticoid killing in vivo could be related to the relative rates of synthesis of a few proteins.