The sera from normal and tumor-bearing rats were separated by polyacrylamide gel disc isoelectric electrophoresis and then in a perpendicular dimension by polyacrylamide gel slab electrophoresis with a gradient from 4% to 20%. When the gel was stained with Coomassie brilliant blue, 100-150 components were separately observable with normal serum. With the sera from Rhodamine sarcoma-bearing rats, three more components were detected with a high reproducibility. They were of pI (isoelectric point) 4.5 and Rm (relative mobility) 0.63, of pI4.8 and Rm 0.63 and of pI 4.9 and Rm 0.75. The 4.5/0.63 (pI/Rm) and the 4.8/0.63 components appeared soon after transplanting the tumor, whereas the 4.9/0.75 component appeared at the late stage of its growth. Upon surgical removal of the grown tumor, the former two components disappeared rapidly, while the latter component disappeared gradually. Similar results were obtained with Yoshida sarcoma, and ascites hepatoma AH66 and AH130. A 3rd polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed that the 4.5/0.63 and the 4.8/0.63 components were each composed of a single polypeptide of approximately 64,000 daltons, and that the 4.9/0.75 component was composed of two different polypeptides of approximately 33,000 daltons and approximately 10,000 daltons. It was confirmed by an immunodiffusion experiment that none of these three proteins was alpha-fetoprotein.