Fine-needle aspiration (FNA) cytology has an established role in the investigation of lymphadenopathy in HIV-infected patients. However, changes in the spectrum of disease have been observed since the introduction of highly active antiretroviral therapy (HAART). The aim of the study was to establish whether FNA cytology remains a useful investigative tool in the post-HAART era and to determine whether the cytology results reflect the changing patterns of disease. Retrospective search of the cytopathology database at University College London Hospitals identified 73 FNA cytology procedures performed in 62 patients between January 1998 and December 2006. FNA cytology showed significant disease in 90% of adequate samples. The most common diagnoses were persistent generalized lymphadenopathy (PGL, 50%), infection (22%) and malignancy (18%). Diagnoses could not be made in 31% of patients because of inadequate sampling. An open lymph node biopsy was subsequently performed in 27% of patients. FNA cytology remains an important initial investigation in the post-HAART era, particularly in the diagnosis of PGL, infection and malignancy. Difficulties in diagnosis of Castleman disease and Hodgkin's lymphoma by FNA cytology are recognized.