The plasma membranes of eukaryotic cells are hypothesised to contain microdomains with distinct lipid and protein composition known as lipid rafts. In T cells, cross-linking of lipid raft components triggers signalling cascades. We show that the T-cell antigen receptor (TCR) and a protein tyrosine kinase, Lck, have a patchy plasma membrane distribution in Jurkat T cells at reduced temperatures, although they have a continuous distribution at physiological temperature (37 degrees C). GM1 displays a patchy distribution at reduced temperature after Triton X-100 extraction. The archetypal non-lipid raft marker, the transferrin receptor, displays a more continuous plasma membrane distribution uncorrelated with that of Lck at 0 degrees C. Cold-induced aggregation of the lipid raft-partitioning proteins is accompanied by increased tyrosine phosphorylation and ERK activation, peaking at 10-20 degrees C. Tyrosine phosphorylation is further greatly increased by ligating the TCR with anti-CD3 at 10-20 degrees C. The tyrosine phosphorylation mainly occurred at the plasma membrane, was dependent on Lck and on the surface expression of the TCR. The activation of tyrosine phosphorylation and ERK by TCR ligation at reduced temperature also occurred in human primary T cells. These results support the concept that lipid rafts can form in membranes of live cells and that their coalescence stimulates signalling.