Bone marrow (BM) cells fractioned in Percoll gradients yield a low-density fraction (Fr3) highly enriched in suppressor activity. Previously, it has been demonstrated that BM associated suppressor activity was mediated by early myeloid cells, through a mechanism dependent on endogenous IFNγ and nitric oxide production after bacterial stimuli, e.g. lipopolysaccharide (LPS). However, the mechanism(s) through which the IFNγ is produced in BM has not yet been fully elucidated. Therefore, in the present study we investigated the involvement of IL-12, IL-18 and IFNβ on the production of IFNγ and nitric oxide in cultures of BM Fr3 cells, and characterized the IFNγ-producing cells, in response to LPS. The results show that both IL-12 and IFNβ, but not IL-18, are involved on IFNγ production. However, only IFNβ appears to be critical on nitric oxide production. Furthermore, we found that cells of the Thy1.2⁺CD3⁺ phenotype produce IFNγ and are tightly involved on nitric oxide production by BM Fr3 cells. In conclusion, IFNβ appears to be critical on IFNγ- and nitric oxide production by BM cells in response to LPS, through a mechanism that is dependent on Thy1.2⁺CD3⁺ IFNγ-producing cells.