The growth of mycobacteria in human macrophages was examined at an ambient concentration of oxygen (5% CO2 and 95% air, corresponding to 20% O2 or 140 mm Hg PO2) and at a concentration corresponding to tissue levels (5% O2 and 5% CO2 in nitrogen balance, corresponding to 36 mm Hg PO2). Compared with the higher PO2 level, macrophages cultivated at lower PO2 level spread more widely and had an increased glycolytic and decreased oxidative metabolism. Upon PMA stimulation, they displayed a better preserved ability to produce superoxide anion and to respond to IFN-gamma priming by increased superoxide anion production. When infected with either Mycobacterium tuberculosis or Mycobacterium avium, macrophages cultured with the lower PO2 level permitted significantly less growth than those cultured at the higher PO2 level. From Day 0 to Day 7, M. tuberculosis grew an average of 0.39 and 1.17 log CFU in macrophages cultured at lower and higher PO2, respectively (p less than 0.0001). From Day 0 to Day 3 of infection, M. avium decreased in macrophages cultured at lower PO2 on average by 0.19 log CFU but grew by 0.34 log CFU in macrophages cultured at higher PO2 (p = 0.0001). Mycobacteria grew equally well in macrophage-free media at either PO2. Crude lymphokines, rIFN-gamma, or rTNF-alpha did not consistently affect the growth of mycobacteria in macrophages at either high or low oxygen conditions. In conclusion, mycobacteria displayed a reduced growth when cultivated in macrophages at a physiologic PO2 that did not reduce the growth of extracellular bacteria. This effect of PO2 on macrophage antimycobacterial power might explain the preferential localization of tuberculous lesions in body areas with high tissue PO2, such as the lung apex.