T he mechanism of tumor-associated hypoglycemia was examined in 11 patients with hepatocellular carcinoma, 6 of whom presented with severe hypoglycemia and 5 in whom plasma glucose was persistently normal. Serum insulin levels in the hypoglycemic patients were low. Although total serum insulin-like growth factor II (IGF-II) levels in both groups of tumor patients were lower than normal, tumor tissue from hypoglycemic patients contained levels of IGF-II mRNA that were 10-20-fold higher than those present in normal liver. IGF-II immunoreactivity consisted in all cases of a mixture of both higher molecular weight forms and material having the character of IGF-II itself. The former comprised a greater proportion of total IGF-II, in patients with hypoglycemia. Studies to characterize the interactions of IGF-II with serum proteins showed that (a) the radiolabeled peptide bound to an approximately 40,000-D protein in sera from both hypoglycemic patients and normal subjects, (b) sera from hypoglycemic patients and normal subjects had similar capacity to bind the radiolabeled peptide, and (c) the apparent affinities of serum binding proteins for IGF-II were the same for both hypoglycemic patients and normal subjects. Whereas, acid extracted, tumor-derived IGF-II immunoreactive peptides with low or intermediate molecular weights bound to serum proteins in a manner indistinguishable from that of IGF-II itself, the highest molecular weight IGF-II immunoreactive peptide exhibited negligible ability to compete for radiolabeled ligand binding to serum proteins. The low affinity of serum binding proteins for this component suggests that high molecular weight IGF-II immunoreactivity might circulate free and be available for interaction with cell-surface receptors.