OBJECTIVE To determine serum and pleural concentrations of tumor necrosis factor alpha (TNF-α) in an experimental model of empyema induced by intrapleural inoculation of Staphylococcus aureus or Streptococcus pneumoniae. METHODS Wistar rats were inoculated with S. aureus (SA group, 17 animals) or S. pneumoniae (SP group, 30 animals). The presence of free fluid or pus in the pleural space was investigated. TNF-α levels >150 pg/ml (minimum detection limit) were determined in pleural fluid and blood. Histopathological examination of pleural tissue was performed to determine the severity of infection. RESULTS Serum TNF-α was >150 pg/ml in nine SA versus 10 SP rats. In pleural fluid, TNF-α was >150 pg/ml in 11 SA versus 19 SP rats. Pleural and serum TNF-α concentrations were significantly different in the SP group (P = 0.035), but not in the SA group (P = 0.727). Pleural TNF-α was similar in both groups (P = 0.92), but serum TNF-α was significantly higher in SA (P = 0.03). Out of 17 SA animals, 1 (5.8%) did not develop empyema, versus 4 (13.3%) out of 30 SP animals. A mild inflammatory response was predominant in both groups, but the inflammatory process was significantly more severe in SP (P = 0.012). However, TNF-α levels were not associated with severity of the inflammatory response. CONCLUSIONS We describe a simple and effective rat model of empyema. TNF-α levels above 150 pg/ml in the pleural fluid are useful to confirm empyema, but cannot predict the severity of the inflammatory response. TNF-α levels below 150 pg/ml are useful to rule out empyema.