The involvement of several cytokines in the antitumor effect induced by intrapleural (i.pl.) injection of heat-killed cells of Lactobacillus casei strain Shirota (LC 9018) in mice was investigated. Injection of LC 9018 i.pl. into Meth A fibrosarcoma (Meth A)-bearing mice not only significantly prolonged the survival of the mice, but also effectively inhibited the accumulation of malignant pleural fluid in the thoracic cavity. In the thoracic cavity of tumor-bearing mice treated with LC 9018, we observed large amounts of several cytokines including interleukin (IL)-1beta, interferon (IFN)-gamma, IL-12 and tumor necrosis factor (TNF)-alpha. Both anti-IFN-gamma and anti-IL-12 monoclonal antibody (mAb) treatments partially diminished the antitumor activity of LC 9018 in vivo, while the treatment of anti-IL-1beta mAb did not influence the survival of the mice. However, anti-TNF-alpha mAb treatment completely abolished the antitumor effect of LC 9018 in vivo, suggesting that in this model LC 9018 has a survival-prolonging effect involving certain cytokines. Moreover, i.pl. injection of mouse recombinant TNF-alpha into Meth A-bearing mice pretreated with anti-TNF-alpha mAb partially restored the survival-enhancing effect of LC 9018. These results led us to conclude that TNF-alpha induced by i.pl. injection of LC 9018 plays an important role in the antitumor effect of LC 9018 in vivo.