Hairy cell leukemia (HCL) is a rare clonal B-cell disorder characterized by a distinctive pattern of infiltration of hairy cells (HCs) in the bone marrow (BM), hepatic sinusoids and splenic red pulp. HCs express a wide spectrum of matrix-binding integrins, which influence their migratory behaviour and subsequent tissue localization. CD44 distribution and staining intensity on HCs from the BM and peripheral blood (PB) were investigated using dual-color flow cytometry. HCs from all cases expressed CD44, but the staining intensity of the positive population was significantly higher than normal residual lymphocytes (p < 0.0005) and medullary HCs in those patients exhibiting additional hairy cell dissemination in the peripheral blood. It is of interest to speculate that the BM microenvironmental may deliver signals which down-regulate CD44 expression on resident HCs compared to HCs recirculating in peripheral blood. CD44 expression on HCs may help clarify their homing mechanisms in the bone marrow and to define phenotypic subsets with different clinical and pathological behaviours.