A large fraction of glycosylphosphatidylinositol (GPI)-anchored proteins and Src-like kinases are confined to glycolipid-enriched membrane (GEM) microdomains. The particular membrane topology of GPI-anchored proteins has led to the postulation of the existence of integral membrane proteins linking extracellular stimuli with cytosolic machinery for endocytosis and signaling. The human MAL cDNA was identified during a search for novel genes differentially expressed during T cell development, and encodes a multispanning membrane protein displaying lipid-like properties. To address the biochemical characterization of endogenous MAL and to analyze its possible association with other proteins, we have generated a monoclonal antibody (mAb) specific to the MAL molecule. Using this mAb, we have identified MAL in GEM microdomains of both the HPB-ALL T cell line and human peripheral blood lymphocytes. Co-immunoprecipitation experiments with antibodies to the MAL molecule or to the GPI-anchored CD59 antigen indicated specific association of MAL with GPI-anchored proteins and Src-like tyrosine kinases. In addition, both MAL and the Src-like kinase Lck were identified in GEM obtained from an endosomal-enriched membrane fraction. These features of MAL closely match some of the properties expected for the hypothetical integral membrane linker proteins acting in specialized GEM-mediated functions.