Selection of the T cell receptor (TCR) repertoire in the thymus probably involves TCR-mediated signals transduced in developing thymocytes after interaction with thymic stromal cells bearing self ligands. TCR-transduced signals should have identifiable consequences that would distinguish thymocytes whose TCRs have been engaged by self ligands from those whose TCRs have not. Among thymocytes expressing a transgenic TCR of defined specificity, a large number had elevated intracellular calcium concentrations but only when resident in a negatively selecting thymus in which their self ligand was expressed. Thus, developing thymocytes are stimulated by endogenous ligands in vivo to mobilize intracellular calcium, and increased intracellular calcium concentrations may reflect the consequences of intrathymic signaling associated with thymic negative selection.