The content of carcinoembryonic antigen (CEA) and its subcellular distribution were studied in nine human colon carcinoma cell lines. A great variation in CEA content was found among different colon cancer cell lines. Well-differentiated colon cancer cell lines (LS174T and SKCO-1) contained the highest CEA activity which was 35 to 60 times greater than that of less well-differentiated cells (SW620, SW480, and HRT18). More than 80% of the CEA was associated with the cell membrane in all nine cell lines. With increasing cell density, the CEA content per cell was found to increase in SW1116, HCT8, and HCT48 cells, while no change was observed in SW620, HRT18, and HT29 cells. In SW480, LS174T, and SKCO-1 cells, CEA content actually decreased with increasing cell density. Investigation of the synthesis of CEA in cells and its release into the medium over an 8-day period showed that the rate of CEA synthesis at maximum cell density for LS174T and SKCO-1 cells decreased to 15 and 50% of that at low cell density, respectively. In contrast, the rate of CEA release into medium by these two cell lines was higher at maximum than at low cell density. For HCT48 cells, the increased rate of CEA synthesis with increasing cell density markedly elevated cellular CEA levels. These observations were confirmed by studying the rate of incorporation of N-acetyl[3H]-glucosamine into cellular CEA in LS174T and HCT48 cell lines. The rate of incorporation of radioactivity into CEA was greater during the exponential phase of growth than during the stationary phase for LS174T, while the opposite was observed with HCT48 cells. This study indicated that there is a great variation in CEA content among different human colon cancer cell lines and that it is associated predominantly with the membrane fraction. The rate of synthesis and release of CEA also varied among different cell lines. The growth phase had a varied effect on the CEA content, the rate of synthesis, and the release of CEA in these human colon cancer cell lines.