TCR modulation induced by anti-TCR or anti-CD3 mAbs leads to a transient state of refractoriness of the T cell to all signals given via cell surface structures. To investigate the underlying mechanisms, we have used human CTL permeabilized with the alpha toxin of S. aureus. This method of permeabilization allows manipulation of the interior milieu of the cell, but maintains its functional and structural integrity. Introduction of the G protein activator GTP gamma S into permeabilized CTL leads to triggering of granule exocytosis. The G protein inactivator GDP beta S inhibited exocytosis induced by TCR triggering but not that induced by activation of protein kinase C. This indicates that the G protein that triggers exocytosis is localized after CD3 triggering but before formation of the polyphosphoinositol breakdown product diacylglycerol. In TCR-modulated CTL, GTP gamma S is no longer able to activate exocytosis. Such CTL, however, still respond to PKC activators. This demonstrates that a TCR-associated G protein has been functionally inactivated by TCR modulation.