Efforts to devise immunoassays for tuberculosis (TB) that can be adapted to rapid formats are ongoing. The present study was aimed at determining whether urinary anti-Mycobacterium tuberculosis antibodies are present in patients with TB, to evaluate the feasibility of developing a urine antibody-based diagnostic test. Urinary antibodies directed against the culture filtrate proteins of M. tuberculosis, MPT 32, and the 81-kDa GlcB protein were detectable in patients with TB, although the sensitivity of antibody detection was lower (53%-64%), compared with serum antibodies (68%-77%). Surprisingly, with all 3 antigens, the use of paired serum and urine samples provided higher sensitivities of antibody detection than either single specimen, and anti-GlcB antibodies were present in the serum and/or urine of 39 (90%) of 43 smear-positive patients with TB. Although, with the current methods and antigens, the level of sensitivity is insufficient to design a urinary antibody diagnostic test, these studies provide the foundation for further studies on the development of a urine antibody-based immunoassay for TB.