Several T cell surface molecules can activate signal transduction pathways that lead to T cell activation. Like the T cell antigen receptor (TCR), several other molecules, including the sheep erythrocyte receptor CD2, are able to activate the phosphatidylinositol (PI) signal transduction pathway upon stimulation with appropriate agonists. However, CD2-initiated activation of this pathway is dependent on the functional expression of the TCR. Since the T cell does not express other known receptors that activate the PI pathway independent of the TCR, the specificity of the CD2 requirement for a functional TCR is not known. To evaluate the specificity of this requirement, we examined the functional capacity of CD2 to activate the PI pathway in a TCR-deficient cell which had been transfected with a heterologous receptor, the human muscarinic subtype 1 receptor (HM1). HM1 is a member of the cholinergic family of receptors and is known to activate the PI pathway. HM1 can function in the absence of the TCR in a Jurkat-derived T cell host. Here we demonstrate through calcium fluorimetry and PI metabolism assays that HM1 is unable to substitute functionally for the TCR in CD2-initiated signal transduction. These results suggest a specific functional interaction between CD2 and the TCR in CD2-mediated activation of the PI pathway in T cells.