Monoclonal antibodies defining B-, T-, and myeloid-restricted cell surface antigens were used to characterize the lineage and state of differentiation of tumor cells isolated from 22 patients with hairy cell leukemia (HCL). These tumors were shown to be of B lineage because they strongly expressed the B cell-restricted antigens B1 and B4 and lacked T cell- and monocyte-restricted antigens. Moreover, the strong expression of the plasma cell-associated PCA-1 antigen on the majority of hairy cells suggested that these tumors correspond to later stages of B cell ontogeny. Dual fluorescence experiments further confirmed that HCL splenocytes that coexpressed B1 and PCA-1 demonstrated both the morphology and tartrate-resistant acid phosphatase positivity of hairy cells. The observation that some hairy cells either spontaneously produce immunoglobulin (Ig) or could be induced to proliferate and secrete Ig provides complementary support for the view that HCL is a pre-plasma cell tumor. However, staining of hairy cells with anti-IL2R1 monoclonal antibody, which is directed to the T cell growth factor receptor and/or with the anti-Mo1 reagent, directed to C3bi complement receptor, distinguish these cells from currently identified B cells.