The current model suggesting that raft integrity is required for T cell activation is mostly (but not exclusively) based on the use of drugs, such as methyl-beta-cyclodextrin (M beta CD), that disorganize rafts and inhibit T cell receptor (TCR)-induced Ca2+ influx. Here we show that conditions that disrupt lipid raft integrity do not inhibit TCR triggering in Jurkat cells and normal T lymphocytes. Indeed, we found that the reported inhibition of TCR-induced Ca2+ influx by M beta CD treatment is mainly due to (a) nonspecific depletion of intracellular Ca2+ stores and (b) plasma membrane depolarization of T cells. When these side-effects are taken into account, raft disorganization does not alter TCR-dependent Ca2+ signaling. In line with these results, also TCR-induced tyrosine phosphorylation is not inhibited by dispersion of lipid rafts. By contrast, in the same conditions, Ca2+ signaling via the glycosylphosphatidylinositol (GPI)-anchored protein CD59 is totally abolished. These results indicate that, while signaling through GPI-anchored proteins requires lipid raft integrity, CD3-dependent TCR activation occurs independently of cholesterol extraction.