Background: Human cases of hypersensitivity pneumonitis (HP) have been reported among machinists for over ten years. Although mycobacteria have been implicated as causal agents, this has not been established in experimental studies and the mechanisms remain unclear. Other constituents of in-use metal working fluids (MWF) may also contribute to the development of lung disease. Thus, we investigated the potential for Mycobacterium immunogenum (MI) in MWF to induce HP. Methods: Mice were exposed intranasally for three weeks to MI (isolated from MWF), Saccharopolyspora rectivirgula (SR) (positive control), saline, endotoxin, MWF spiked with endotoxin and/or MI, used MWF and particulate-fortified used MWF. Responses were assessed 96 hrs after the last exposure. Results: Mice exposed to MI in MWF developed lung pathology consistent with HP along with significantly more monocytes and neutrophils in lung lavage; increased CD4⁺/CD8⁺ T-lymphocyte ratio; and marked pulmonary lymphocytosis on histological exam when compared to saline-treated controls. Mice with grade 2 or higher pathology (0-4 point scale) exhibited significantly elevated MIP-1, IL-10 and a trend toward higher RANTES, 96 hrs after the final dose. Endotoxin co-exposure augmented lung pathology. Conclusion: MWF containing mycobacteria induced granulomatous lung lesions, peribronchiolar lymphocytosis, increased cell concentrations in lavage, and upregulation of several cytokines, findings consistent with HP.