CD9 is a member of the tetraspanin superfamily which is characterized by four transmembrane (TM) domains and associates with other surface molecules. This tetraspanin was recently found to be expressed on mature T cells. Here, we investigated which molecules associate with CD9 on T cells and which CD9 domains are required for the association. Immunoprecipitation of T cell lysates with anti-CD9 mAb followed by immunoblotting with mAb against various T cell molecules showed the association of CD9 with CD3, CD4, CD5, CD2, CD29 and CD44. Because association with CD5 was most prominent, we determined the role of CD9 TM or extracellular (EC) domains in the association with CD5. CD9 mutant genes lacking each domain were constructed and introduced into EL4 thymoma cells deficient in CD9 but expressing CD5. Among various types of stable EL4 transfectants, EL4 transfected with the mutant gene lacking TM domains (TM2/TM3) between two EC domains expressed a small amount of the relevant protein without showing association with CD5. CD9(-)CD5(-) monkey COS-7 cells transfected with this mutant gene and the CD5 gene expressed both transfected gene products, but the association of these was not detected. EL4 cells transfected with a CD9/CD81 chimera gene (the CD9 gene containing TM2/TM3 of CD81) expressed the chimeric protein on the cell surface and showed association with CD5. These results suggest an essential role of particular CD9 TM domains in the surface expression of the CD9 molecule as well as the association with CD5.