||The adaptive immune response is essential for elimination of invading pathogens that have escaped innate immunity. The activation of CD4 + T cells, a major cell population of adaptive immunity, requires many known molecular interactions, although the spatial organization of such signaling molecules is unclear. It has been hypothesized that signaling molecules are associated with domains of the membrane enriched in sphingolipid, cholesterol, and glycosylphosphatidyl-inositol (GPI)-anchored proteins, known as membrane rafts. Previous studies examining membrane rafts have used a detergent-based membrane raft isolation protocol; however, the use of a detergent-based isolation protocol has been questioned. Therefore, we have developed a detergent-free protocol to isolate membrane rafts from CD4 + T cells. We found two subsets of membrane rafts and determined their size. We also isolated membrane rafts from stimulated T cells and determined their size. These data could provide further insight into understanding the mechanics of T cell activation.