For peripheral T-cell lymphomas (PTCL), anti-CD52 (alemtuzumab) therapy is currently under investigation. Previous studies reported widely divergent CD52 expression within PTCL using conventional immunohistochemistry. To accurately discriminate between the presumed mechanistically relevant CD52 expression in tumor versus bystander cells, we employed immunofluorescence double stains using CD52 in combination with an antibody directed against the rearranged T-cell receptor Vβ-segment of the neoplastic clone in 6 angioimmunoblastic T-cell lymphomas (AITL) and 5 PTCL, unspecified (PTCL-NOS) and, in combination with CD30, in 18 anaplastic large cell lymphomas (ALCL). Tumor cells in all AITL and PTCL-NOS were CD52 positive, while in 17 of 18 ALCL no specific staining was observed. Conversely, the background T- and B-cell infiltrate showed a consistent positivity for CD52. Our approach helps to precisely define CD52 expression in the tumor cell population of PTCL and might therefore be valuable when evaluating the response to alemtuzumab therapy in prospective clinical trials.