Tuberculosis (TB) is one of the oldest human infectious diseases and one third of the world's population is latently infected. Brazil is an endemic area for TB. One of the most important challenges in TB control is the identification of latently infected individuals. Health Care Workers (HCW) are at high risk of being infected with Mycobacterium tuberculosis and even to become TB latently infected. The aim of this study was to increase knowledge about humoral immune response in TB latently infected individuals. HCW were classified according to their tuberculin skin test (TST), as positive or negative. The antibody response to GLcB, MPT51 and HSPX from Mycobacterium tuberculosis was evaluated. TST negative HCW constituted the majority of those who showed a humoral immune response. Antibody levels varied according to antigen characteristics, TST and BCG status. We suggest that possibly the presence of those antibodies could have a function in the protective immune response against Mycobacterium tuberculosis.