Oligosaccharides with Le^x determinant (Galβ1→4[Fucα1→3]GlcNAc) are accumulated in large quantities in various adenocarcinomas. Monoclonal antibodies recognizing mono-, di-, or trimeric Le^x showed a preferential staining of specific stages of human fetal tissues and various human adenocarcinomas. Thus, these carbohydrate epitopes are typical of oncodevelopmental antigens. The present study investigated the presence of Le^x epitope in sera of normal individuals and cancer patients, utilizing two high-affinity monoclonal antibodies, SH1 and SH2, directed to mono- and dimeric Le^x structures, respectively. The Le^x antigen in serum was eluted in the void volume fraction of a gel filtration column, determined by using monoclonal antibody SH1, and found to be carried on a glycoprotein with a molecular weight of approximately 200,000. The Le^x antigen was present in the void volume fraction of the majority (85%) of sera from adenocarcinoma patients. Although the Le^x epitope was also detected in a smaller proportion (33%) of normal sera, its levels were significantly lower than in cancer sera. Le^x antigen was also detected in serum glycolipid fraction; however, no significant differences were observed in normal and cancer sera. A double determinant solid phase immunoassay utilizing SH2 as the capture antibody and SH1 as the detecting antibody allowed direct determination of Le^x levels in sera. By the use of this direct assay, the levels of serum Le^x were found to increase in association with the progression of colorectal cancer (Dukes A to D). The percentage of detectability in sera from colon cancer patients was as follows: Dukes A, 20%; Dukes B, 45%; Dukes C, 67%; and Dukes D, 74%. The levels of serum Le^x were also of prognostic value in Dukes C cancer patients after surgery and during postoperative follow-up.