In T cells, the lipid raft-associated Lck is strongly tyrosine phosphorylated and has reduced enzymic activity in contrast with the detergent-soluble pool, which has substantial activity. Lck tagged at the C-terminus (Lck/V5-His) was efficiently captured by epitope-specific reagents from the detergent-soluble fraction but not from lipid rafts. Binding was restored following urea denaturation, suggesting that Lck/V5-His is in a ‘closed’ conformation in these domains. In agreement with this hypothesis, the Tyr⁵⁰⁵ → Phe/V5-His and Arg¹⁵⁴ → Lys/V5-His mutants, which disrupt the SH2-Tyr⁵⁰⁵ intramolecular interaction, were efficiently precipitated from lipid rafts. In contrast to Lck, Fyn/V5-His was precipitated equally well from both fractions. In the LAT (linker of activated T cells)-deficient J.CaM2 cells, Tyr⁵⁰⁵ phosphorylation of raft-associated Lck was reduced whereas its enzymic activity was elevated. This correlated with decreased levels of raft-localized Csk (C-terminal Src kinase) kinase. Increased tyrosine phosphorylation of Lck was restored in LAT-reconstituted J.CaM2 cells suggesting that LAT negatively regulates Lck activity in lipid rafts. Co-immunoprecipitation experiments from Tyr⁵⁰⁵ → Phe/V5-His-expressing cells revealed that LAT preferentially interacts with the ‘open’ form of Lck in T cell raft domains. These results demonstrate that, unlike the non-raft pool, Lck in lipid rafts has a ‘closed’-inactive structure, and that LAT plays a role in maintaining this conformation, possibly by facilitating critical associations within lipid rafts via its capacity to interact with the ‘open’ form of the kinase.