TNFα is a potent pro-inflammatory cytokine produced by macrophages and is thought to play a critical role in development of chronic lung disease in neonates. Respiratory tract colonization with mycoplasmas is associated with chronic lung disease in neonates presumably by increasing inflammation. Surfactant has been known to have immunomodulatory effect on alveolar macrophages. We hypothesized that surfactant would reduce TNFα production in murine macrophages stimulated by genital mycoplasmas. RAW 264.7, a murine macrophage cell line, was pre-incubated with surfactant (0%,15%, 50%) for 2 hours and then stimulated with Mycoplasma hominis and Ureaplasma urealyticum (104, 106 cfu/ml) and allowed to incubate for 16 hours in 5% CO2. LPS (10 ng, 100 ng) and sterile media were used as controls. A similar set of experiments were run in either 21% O2 and 5%CO2 or 95% O2 and 5% CO2. Cells were incubated for 16 hours and supernatants were frozen at-70°C until assayed for TNFα by ELISA (Endogen Inc.). We found that surfactant produced a concentration-dependent suppression of LPS-induced TNFα production. M. hominis and U. urealyticum also induced the cells to produce TNFα which was dependent on the concentration of the organisms. A dose-dependent suppression of TNFα was seen with LPS and M. hominis, whereas suppression was only seen with the highest doses of surfactant with U. urealyticum. Pre-incubation with IFNγ was synergistic and this synergism was significantly suppressed by higher concentration of surfactant (50%). Hyperoxia (95%O2) produced little or no amplification of TNFα and surfactant suppressed the production as in normoxia (21% O2). We conclude that M. hominis and U. urealyticum are potent inducers of TNFα from the murine macrophages and that surfactant inhibits the response in a dose-dependent fashion with LPS and M. hominis, but only the higher concentration of surfactant suppressed the response to U. urealyticum and this inhibition was also seen in hyperoxia.