BACKGROUND Anaplastic large cell lymphoma (ALCL) is usually diagnosed by histologic and immunohistochemical analysis. However, fine-needle aspiration is becoming a popular alternative to lymph node biopsy, and flow-cytometric immunophenotyping is often used to analyze fine-needle aspiration specimens. OBJECTIVE To review our experience using flow-cytometric immunophenotyping to assess cases of ALCL and to evaluate the diagnostic utility of this technique. METHODS Each case of ALCL was assessed by flow cytometry with 3-color or 4-color antibody panels, and data were reanalyzed by cluster analysis using Paint-a-Gate for cases with retrievable flow cytometry data files. Anaplastic lymphoma kinase (ALK) was assessed by using immunohistochemistry. RESULTS Twenty-three ALCL cases were analyzed by flow cytometry. In 4 cases, neoplastic cells could not be identified. In the remaining 19 cases (11 ALK(+), 8 ALK(-)), all were positive for CD30 and CD45. Anaplastic large cell lymphoma cells were large and usually CD45 bright, with many or most cells falling in the region of monocytes on the CD45/side scatter plot. The frequencies of T-cell antigen expression in ALK(+) cases were CD2, 67%; CD7, 60%; CD3, 45%; CD4, 33%; CD5, 14%; and CD8, 14%. In ALK(-) cases, the frequencies of the T-cell antigen expression were CD2, 100%; CD3, 50%; CD4, 40%; CD7, 40%; CD5, 25%; and CD8, 20%. CONCLUSIONS Flow cytometry can be used to immunophenotype ALCL cases. Neoplastic cells may be few, and they may fall outside of the lymphocyte gate. Cluster analysis using software like Paint-A-Gate is often helpful because it allows for flexible, sequential gating strategies to identify and characterize the neoplastic cells.