Tumor necrosis factor-α (TNF-α) is a proinflammatory cytokine that appears to play a significant role in the development of neonatal chronic lung disease (CLD). Inflammation and CLD are also associated with respiratory tract colonization with genital mycoplasmas. The possible protective roles of surfactant in mitigating the inflammatory response to these microbes were investigated. Murine RAW 264.7 macrophages were preincubated with an exogenous surfactant and exposed overnight to sterile media, lipopolysaccharide (LPS), Mycoplasma hominis, or Ureaplasma urealyticum. Macrophages released TNF-α in response to challenge with LPS, U. urealyticum, and M. hominis in a concentration-dependent fashion. Surfactant suppressed LPS and M. hominis induced TNF-α production in a dose-dependent manner but suppressed U. urealyticum-mediated TNF-α production only at the higher dose tested. Similar effects were seen in hyperoxia (95% O2). Thus, exogenous bovine surfactant significantly inhibits the production of TNF-α by murine macrophages stimulated with genital mycoplasmas and bacterial LPS.