Pertussis toxin is known to elicit lymphocytosis in whooping cough patients and experimental animals, by blocking the extravasation of lymphocytes and stimulating their release from lymphoid organs such as the thymus. The mechanisms responsible for these unique effects of PT are not fully understood. The effect of pertussis toxin (PT) on the invasive behavior of human CCRF-CEM T lymphoma cells has been investigated with the use of a monolayer invasion assay (MIA). We had previously found that invasion of murine T lymphoma cells in this model system was correlated with their ability to extravasate and form metastases after i.v. injection in syngeneic animals. We now show that human CEM cells can also penetrate through a precultured confluent monolayer of murine 10T1/2 fibroblast-like cells within a few hours. In a quantitative MIA run over 24 h, PT at concentrations above 10(-14) M inhibited invasion of the CEM cells. In addition, PT stimulated the release ('evasion') of CEM cells that had invaded under the monolayer before the toxin was added. The A subunit of PT was totally inactive, the B subunit had a small residual effect, and reconstitution of the AB complex partially restored the activity. The invasion-inhibiting activity of two different holotoxin preparations and of the subunits perfectly matched their activity in the Chinese hamster ovary cell clustering assay, which is known to depend on a functional AB complex. We suggest that inhibition of monolayer invasion by PT can be used as an in vitro model system to investigate the cellular and molecular mechanisms underlying the lymphocytosis-promoting action of the toxin. Furthermore, the method is sufficiently sensitive to be used for titration of toxin activity. Our data indicate that the ADP-ribosylating activity of the A subunit is indeed required, and that the promotion of lymphocytosis is not elicited by the binding of the B subunit alone.