The binding of pertussis toxin (PT) to the human T-cell line Jurkat was examined by using flow cytometry. Fluorescein isothiocyanate (FITC)-labeled PT bound rapidly to the cells in a specific manner as determined by blocking experiments with unlabeled toxin, B oligomer, and the S2-S4 and S3-S4 dimers. Monoclonal antibodies against the S3 subunit of the toxin also significantly inhibited the binding of FITC-PT. Sialidase treatment of the cells resulted in decreased binding of FITC-PT, indicating that sialic acid residues are involved in the binding process. In addition, we studied the effect of PT binding on the expression of cell surface molecules. On binding of PT to the cell surface, a rapid down-regulation of the T-cell receptor (TCR)-CD3 complex was observed. The modulation of the TCR-CD3 complex was independent of the toxin's enzymatic activity, as the B oligomer and a nonenzymatic toxin mutant induced modulation comparable to that caused by the native holotoxin. Isolated dimers did not cause down-regulation. Stimulation of the TCR-CD3 complex, leading to reduced cell surface expression of this complex, provides a possible explanation for the second messenger production associated with the interaction of PT or B oligomer with T lymphocytes. We therefore conclude that PT activates T cells by divalent binding to the TCR-CD3 complex itself or by binding a structure closely associated with it.