Providing prompt and precise laboratory confirmation of a clinical diagnosis of tuberculous lymphadenitis is difficult given the paucibacillary nature of lymph node specimens. In this study carried out in Karachi, Pakistan, a polymerase chain reaction (PCR) assay aimed at detecting Mycobacterium tuberculosis DNA in peripheral blood mononuclear cells (PBMC-PCR) was standardized and compared with standard M. tuberculosis diagnostic techniques or a lymph node PCR (LN-PCR) for the diagnosis of tuberculosis lymphadenitis. Thirty-seven (77%) specimens from 48 patients with clinical or diagnosis of tuberculosis lymphadenitis were positive by cytology [17/48 (35%) with no acid fast bacilli (AFB) (suggestive), and 20/48 (42%) with AFB (positive) in direct smears], 30 (63%) by PBMC-PCR, 16 (33%) by LN-PCR, and 13 (27%) by culture. All controls were negative, with the exception of one false-positive LN-PCR. These data suggest the PBMC-PCR may be helpful in the diagnosis of tuberculous lymphadenitis.