Activated mast cells reside in close apposition to T cells in some inflammatory processes. In this study, we analyzed whether this close physical proximity affects human mast cell degranulation and cytokine release. Thus HMC-1 human mast cells or primary bone marrow-derived human mast cells were cocultured with activated and with resting T cells. Mast cells cocultured with activated T cells released histamine and ␤-hexosaminidase and produced tumor necrosis factor ␣ (TNF-␣), an effect that peaked at 20 h. Kinetics of histamine release paralleled the formation of heterotypic aggregates. Separation of the two cell populations with a porous membrane prevented mediator release and TNF-␣ production. Addition of the PI3-kinase inhibitor, wortmannin, inhibited the heterotypic adhesion-associated degranulation but not TNF-␣ production. These data thus indicate a novel pathway through which human mast cells are activated to both release granule-associated mediators and to produce cytokines in association with heterotypic adhesion to activated human T cells.