The study of lipid microdomains in the plasma membrane is a topic of recent interest in leukocyte biology. Many T cell activation and signaling molecules are found to be associated with lipid microdomains and have been implicated in normal T cell function. It has been proposed that lipid microdomains with their associated molecules move by lateral diffusion to areas of cellular interactions to initiate signaling pathways. Using sucrose density gradients we have found that human T cell beta(1) integrins are not normally associated with lipid microdomains. However, cross-linking of GM1 through cholera toxin B-subunit (CTB) causes an enrichment of beta(1) integrins in microdomain fractions, suggesting that cross-linking lipid microdomains causes a reorganization of molecular associations. Fluorescent microscopy was used to examine the localization of various lymphocyte surface molecules before and after lipid microdomain cross-linking. Lymphocytes treated with FITC-CTB reveal an endocytic vesicle that is enriched in TCR and CD59, while beta(1) integrin, CD43, and LFA-3 were not localized in the vesicle. However, when anti-CTB Abs are used to cross-link lipid microdomains, the microdomains are not internalized but are clustered on the cell surface. In this study, CD59, CD43, and beta(1) integrin are all seen to colocalize in a new lipid microdomain from which LFA-3 remains excluded and the TCR is now dissociated. These findings show that cross-linking lipid microdomains can cause a dynamic rearrangement of the normal order of T lymphocyte microdomains into an organization where novel associations are created and signaling pathways may be initiated.