Non-retractable cell surface projections and cytoskeleton-mediated functional defects are distinguishing features of both hairy cell leukemia (HCL) and neutrophil actin dysfunction (NAD). These defects in NAD neutrophils are attributed to moderate over-expression of pp52 (LSP1), the F-actin-binding, leukocyte-specific phosphoprotein. Here we report that pp52 is similarly elevated in HCL patient PBMCs. Established HCL cell lines exhibited characteristic morphological features like those of fresh HCL cells and showed elevated pp52 levels. The excess pp52 in these HCL cell lines was selectively associated with the F-actin-rich cytoskeletal arrays in surface projections. Treatments producing radical changes in HCL cell shape also altered pp52 expression and intracellular distribution. Alpha interferon (IFNalpha, used to treat HCL) reduced pp52 levels, normalized intracellular pp52 distribution and reverted HCL cells to rounded B cell morphology. Phorbol ester stimulation rapidly generated hyper-phosphorylated pp52 isoforms which translocated from the cytoskeleton to the cytosol prior to the further elongation of surface spikes. This indicates a direct role for phosphorylation in controlling pp52 interactions with the cytoskeleton. Overall, these findings strongly suggest that elevated pp52 expression and/or selective cytoskeletal association contributes to the distinctive morphology of HCL cells.