We found that in vitro multinucleated giant cells (MGC) could be produced by incubation of highly purified human blood monocytes with Con A alone, and the effect of Con A was dose- and time-dependent. Any of the cytokines considered as macrophage-activating factor, such as IFN-gamma, IL-2, IL-4, GM-CSF, or TNF-alpha, did not induce MGC by itself. When added to monocyte cultures, however, IFN-gamma enhanced Con A-induced MGC formation in a dose-dependent manner. In contrast, IL-4 suppressed this response in a dose- and time-dependent manner. IL-4 antagonized the enhancing effect of IFN-gamma on Con A-induced MGC formation. This ability to suppress the formation of MGC was completely abrogated after treatment with anti-IL-4 antibody. In addition, the involvement of monokines (IL-1 alpha, IL-1 beta, IL-6, and TNF-alpha) in Con A-induced monocyte fusion was investigated by adding various antimonokine polyclonal antibodies in cultures. Normal rabbit IgG, anti-IL-1 alpha rabbit antibody, and anti-IL-1 beta rabbit antibody had no effect on Con A-induced MGC formation. However, anti-TNF-alpha rabbit antibody had suppressed the monocyte fusion induced by Con A in a dose-dependent manner, and a high dose of anti-IL-6 rabbit antibody had a partially suppressive effect. Anti-TNF-alpha mAb also had an inhibitory effect on monocyte fusion. Furthermore, the enhancing effect of IFN-gamma on this response was entirely abrogated by anti-TNF-alpha rabbit antibody. There was a highly significant positive correlation between the fusion rates of monocytes and the levels of TNF-alpha produced by monocytes (r = 0.68, p < 0.0005). Our results indicate that T cell-derived lymphokines, such as IFN-gamma and IL-4, have mutually antagonistic effects on Con A-induced human MGC formations in vitro, in which the expression of TNF-alpha in human monocytes plays a key role in the fusion process of monocytes.