Despite its usefulness in the diagnosis of tuberculous lymphadenitis, fine needle aspiration cytology (FNAC) faces several limitations, and its sensitivity and specificity are not well established. The diagnostic accuracy and limitations of FNAC were studied in comparison with conventional microbiological methods and polymerase chain reaction (PCR). Sixty patients with lymphadenopathy and a clinical diagnosis of tuberculous lymphadenitis were subjected to FNA. The aspirate was used for cytological examination, Ziehl-Neelsen staining, mycobacterial culture and PCR. PCR was performed using two sets of oligonucleotide primers for Mycobacterium tuberculosis and a single primer for M. bovis species. The results of FNAC, microbiological methods and PCR correlated with the clinical outcome after follow-up for an average period of 24 months. Twenty-five cases (41.6%) were treated and responded well to anti-tuberculosis therapy, among them 17 were correctly diagnosed by FNAC (68%), eight by microbiological methods (32%) and 24 by PCR (96%). When PCR is considered the gold standard, FNAC predicted the correct diagnosis in 62% of cases with a high false negative rate (38%) due to the absence of granuloma/necrosis in smears from cases of early tuberculosis. In the latter group PCR proved to be the most valuable and a diagnostic success of 100% was achieved when FNAC and PCR were combined. In addition, PCR allowed immediate characterization of M. tuberculosis in the vast majority (96.2%) of cases in the study population.