The cluster designation (CD)4 molecule is one of several nonpolymorphic T lymphocyte surface proteins that have been implicated in T cell-target cell interactions, and is thought to play an important role in regulating T helper cell function. Previously, we found that gangliosides inhibited the function of rat T helper cell lines, and simultaneously inhibited the expression of the rat CD4 molecule identified by the W3/25 antibody. We have now evaluated the generality and mechanism(s) of ganglioside-induced modulation of CD4 expressed by mouse, rat, and human T helper lymphocytes. Ganglioside pretreatment induced rapid and selective disappearance of the CD4 molecule from T helper cells of all three species. The ganglioside effect was temperature- and dose-dependent, reversible within 24 hr of ganglioside removal, azide-insensitive, and was neutralized completely by 10% serum. CD4 modulation appeared to be a general property of gangliosides since the effect could be induced similarly by highly purified individual gangliosides with varying amounts of sialic acid, or by mixed gangliosides. The activity of gangliosides appeared to require both the lipid and sialated oligosaccharide moieties. Gangliosides did not inactivate antibody function, but prevented binding at the cell surface by 12 different monoclonal antibodies specific for a variety of different CD4 epitopes. Preclearance of CD4 by antibody-mediated capping reduced binding of 3H-GM1 to T helper cells. Labeled GM1 bound to several detergent-extracted and transblotted lymphocyte-associated proteins, but apparently did not bind directly to the CD4 molecule under these conditions. These results indicate that gangliosides induce a profound change in the molecular orientation of CD4 within the T helper cell membrane which renders epitopes on the CD4 molecule inaccessible to antibody. This ganglioside effect represents a novel pathway which may contribute to the understanding of the role of CD4 as a regulatory molecule and as a specific receptor for the acquired immune deficiency syndrome virus.