Background: Latent tuberculosis infection (LTBI) is caused by Mycobacterium tuberculosis (MTB) in a state of nonreplicating persistence (NRP). Recent evidence suggests that some very specific adaptations to oxygen depletion occur so that MTB undergoes hypoxic NRP state. In this study the modified slowly stirred, limited Head Space Ratio (0.5HSR) method was used to investigate the physiological response of MTB to different oxygen tension levels. Materials and Methods: For setting up the various NRP stages, some susceptible and drug resistant clinically isolated strains of MTB were cultivated in Dubos Tween-Albumin medium via hypoxically, slow stirring 0.5 HSR methods. Additionally, the effects of isoniazid, rifampin, pyrazinamide, ciprofloxacin and metronidazole against MTB were examined during NRP-1 and NRP-2 stages. The α-crystalline protein was detected during NRP-1 stage of the MTB cultures via performance of the suitable procedures for pellet preparation, washing and cell disruption and SDS-PAGE(Sodium dodecyl sulfatepolyacrylamide gel electrophoresis) technique. Results: NRP-1 and NRP-2 stages of MTB were assessed. The first three of the four drugs mentioned above affected the MTB at actively replicating period and the rifampin effect was continued slightly during NRP-1 stage. Metronidazole affected the MTB at anaerobic NRP-2 stage. Alpha-crystalline protein was detected in NRP -1 stage but was not detected at aerated cultures. Conclusion: Induction of the α-crystalline protein during hypoxic shift-down of MTB metabolism, and its function as a chaperone, suggests a critical role for this protein in the ability of MTB to persist without replicating in the hostile regions of the host's tissues. Therefore, for an effective TB control program, it is critical to understand the mechanisms of factors induction associated with the hypoxic condition of tubercle bacilli and some strategies for the identification of new drug targets must be developed and the persistence states in human lesions should be prevented as well. (Tanaffos 2005; 4(14): 43-52)