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Notes: To explain the mechanism of inhibition of human lymphocyte transformation by human alpha-fetoprotein (HAFP), we sought for, and failed to find, evidence of physical association between HAFP and phytomitogens or antihuman thymocyte antiserum. In addition, 20–40 fold increases in mitogen dose do not reverse the inhibition of lymphocyte transformation by a constant dose of HAFP. The presence of HAFP does not interfere with the attachment of 125I-labelled phytohemagglutinin to the lymphocyte surface. Two-dimensional crossed immunoelectrophoresis of HAFP isolated from the body fluids of hepatoma patients demonstrates three charged species of HAFP designated as HAFP-1 (the most cathodal), HAFP-2, and HAFP-3 (the most anodal). The potency of HAFP isolates in inhibiting lymphocyte transformation can be positively correlated with the ratio HAFP-3: HAFP-1 in each preparation. Passage of HAFP isolates over CM-cellulose allows the isolation of two HAFP fractions, one at pH 4.95, and the other at pH 5.24. The pH 4.95 CM-cellulose isolate is enriched in the more electronegative HAFP species (HAFP-3) and is, on the average, two times more potent than the pH 5.24 CM-cellulose HAFP isolate. The latter, by comparison with native HAFP, is enriched in the more electropositive HAFP species (HAFP-1). The CM-cellulose HAFP isolates are identical in sialic acid content. We have isolated HAFP from the serum, ascitic fluid, and saline extract of tumor from a single hepatoma patient, and from an homogenate of fetal livers. When tested for their capacity to inhibit human lymphocyte transformation in vitro, tumor and fetal liver HAFP were found to be extremely potent; serum HAFP had intermediate potency, and ascitic fluid HAFP was the least potent. Analysis of these HAFP isolates by isoelectric focusing in polyacrylamide gels containing 8 M urea revealed at least 6 molecular HAFP variants. The proportion of one of these variants, termed HAFP-3a, in each isolate was correlated with the immunosuppressive potency of the isolate. The sialic acid content of the various HAFP isolates did not vary significantly. Our data suggest that a post-synthetic modification of HAFP occurs, which modulates its immunosuppressive potency.