AMs from young Wistar rats were obtained by bronchoalveolar lavage and co-cultured with Mycobacterium phlei (MOI 10). We exposed AM cultures to ROFA to characterize the effect of low ROFA concentrations (0, 2.5, and 5 ¦Ìg/ml) and evaluated the response of pre-exposed AM against the bacilli. Low ROFA concentrations induced superoxide anion and nitrites production (p < 0.001). Pre-exposure to ROFA (2.5 and 5 ¦Ìg/ml) caused a significant reduction on TNF¦Á (p < 0.001) and superoxide anion (p < 0.001) production but, did not modify the nitrite production when AM were co-cultured with M. phlei. In addition, ROFA significantly diminished AM killing ability in culture (p < 0.001). Hence, our results indicate that pre-exposure to low levels of ROFA modifies the innate pulmonary defence mechanisms against environmental mycobacteria.