Gangliosides have long been implicated in T cell activation. GD1c with two N-glycolylneuraminic acids [GD1c (NeuGc, NeuGc)] is the predominant ganglioside in rat T cells. In the present study, the anti-GD1c (NeuGc, NeuGc) mAb, AC1, which binds to the NeuGcα2-8Neu-Gcα2- sequence, was found to enhance Con A-activated cellular proliferation at a concentration at which AC1 alone did not activate the cells. The potentiation by AC1 was observed more consistently and effectively in the cellular activation elicited by cross-linking of anti-CD3 and anti-CD4, rather than in the cell growth induced by immobilized anti-CD3 alone. Moreover, the combination of immobilized anti-CD4 and soluble AC1 had a remarkable mitogenic effect. In addition, we have demonstrated the existence of a 100 kDa protein in rat T cell lysates which reacts with AC1 on Western blots, and this interaction is abolished by sialidase-treatment of the membrane. Pronase treatment of the T cells, which rendered the 100 kDa protein undetectable on Western-blotting, reduced the number of AC1-positive cells by 40-50% on flow cytometry. On the other hand, all cells became AC1-negative after sialidase treatment. These findings indicated that AC1 reacts with both GD1c (NeuGc, NeuGc) and the 100 kDa glycoprotein on rat T cells. Taken together, these results predict the presence of a novel regulatory mechanism of T cell activation involving CD4 and the NeuGcα2-8NeuGcα2- sequence.