The immunophenotype of hairy cell leukemia (HCL) was investigated using 20 routinely fixed paraffin-embedded tissue sections (12 bone marrows, six spleens, one liver, one lymph node) from 12 patients known to have this disease. A panel of antibodies was used, including anti-leukocyte common antigen (anti-LCA), B-lineage antibodies (LN2, MB2, L26), T-lineage reagents (MT1, UCHL1), monocytic (anti-cathepsin B) and myelomonocytic (anti-lysosyme, Mac 387) antibodies, and other less lineage-specific markers (anti-S-100, anti-alpha-1-antichymotrypsin (anti-alpha 1-ACT), anti-alpha 1-antitrypsin (anti-alpha 1-AT), anti-vimentin). Anti-LCA stained hairy cells in seven of the 12 bone marrows and consistently recognized hairy cells in the spleen, liver, and lymph nodes. Hairy cells generally reacted with B-lineage antibodies and were not labeled by T-lineage markers. No reactivity was noted with myelomonocytic antibodies, anti-S-100, anti-alpha 1-ACT, or anti-alpha 1-AT. Vimentin was expressed in the majority of cases. Tartrate-resistant acid phosphatase reactivity was demonstrated in three of the 20 routinely processed tissue sections. These data suggest that immunohistochemical studies of hairy cell leukemia in routinely processed tissue may be useful in diagnostic hematopathology and surgical pathology.