Background T-SPOT.TB is a novel test for tuberculosis infection with higher sensitivity and specificity than the traditional tuberculin skin test (TST). However, there are no longitudinal data in the literature evaluating T-SPOT.TB for Mycobacterium tuberculosis in patients with acquired immune deficiency syndrome (AIDS) on highly active antiretroviral therapy (HAART). The objective of this study was to assess the value of T-SPOT.TB longitudinally in AIDS patients on HAART without prophylaxis for tuberculosis.Methods A prospective observational study was conducted in 50 AIDS patients on HAART. None of the subjects had evidence of active tuberculosis. T-SPOT.TB, a T-cell-based interferon y released assay, was performed at the onset of the study and repeated 24 months thereafter. Subjects were evaluated every 6 months during the 36-month follow-up. Results Twenty-one (42%) AIDS patients on HAART tested positive by T-SPOT.TB (95% Cl 28.3%-55.7%). The pooled spot-forming cells of early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) peptides were 68/million peripheral blood mononuclear cell (PBMC) (interquartile range 44-220). The average number of CD4 cells in subjects was (305±152) cells/Ml and there was no significant difference in T-SPOT.TB response rates between subjects with CD4 cell counts <200 cells/ul (7/15 (46.7%), 95% C/21.5%-71.9%) and those with CD4 cell counts≥200 cells/ul (14/35 (40.0%), 95% Cl 23.8%-56.2%, P=0.662). In the 32 subjects who completed the 24-month follow-up, 10 underwent T-SPOT.TB reversion, one had T-SPOT.TB conversion, six remained positive and 15 remained negative. None of them advanced to active tuberculosis during the 36-month follow-up.Conclusion The inactive status of tuberculosis infection may be maintained for a long period in AIDS patients on HAART.