Cells of the immune system tightly regulate the binding ability of cell adhesion molecules. The binding of the extracellular matrix component hyaluronan to CD44 is no exception, yet the mechanisms that regulate its binding are poorly understood. In this study a chimeric CD4/CD44 molecule, containing the extracellular domain of CD4 and the transmembrane and cytoplasmic domains of CD44, was expressed in two CD44+ mouse T lymphoma cell lines, BW5147 and T28. This resulted in the reduced ability of endogenous CD44 to constitutively bind hyaluronan. Immunoprecipitation of the chimeric protein in 1 % Brij-96 indicated an association between the chimera and endogenous CD44. Using various chimeric CD4/CD44 molecules, the transmembrane region of CD44 was found to mediate this association. In addition, the association of chimeric CD4/CD44 molecules with endogenous CD44 correlated with reduced hyaluronan binding. Thus, the transmembrane region of CD44 is required for the association with CD44 molecules in the cell membrane and we propose that the self-association of CD44 molecules occurs on the T cell surface to promote hyaluronan binding. Cellular events altering the interactions of the transmembrane region of CD44 thus have the potential to regulate the hyaluronan binding ability of CD44.