The glycosylphosphatidylinositol (GPI)-anchored protein CD59 and the ganglioside GM1 are present on lipid rafts that can be isolated in a detergent-insoluble membrane (DIM) fraction. TCR engagement promotes integration of components of the TCR/CD3 signaling machinery into DIM. As DIM are isolated as a heterogeneous mixture of coalescent membranes, it is uncertain whether the cofractionation of GPI-anchored proteins and GM1 reflects the existence of an association between these molecules within the same lipid rafts in the cell. We have studied the surface distribution of the co-stimulatory CD59 and GM1 molecules and their role in the recruitment of components of the TCR signaling machinery in DIM. Although both CD59 and GM1 are present in rafts, these molecules occur in a steady state, mainly clustered in different membrane subdomains. Multimerization of either molecule did not induce cocapping or co-internalization of the other. Aggregation of GM1, CD59 or TCR/CD3 increased tyrosine phosphorylation but only in the latter case was a significant increase observed in both tyrosine phosphorylation and recruitment of elements of the signaling machinery in DIM. Our results show the existence of specific co-stimulatory membrane microdomains that require a direct TCR/CD3 engagement for efficient recruitment of signaling machinery into rafts.