Infection with Mycobacterium bovis bacillus Calmette-Guerin (BCG) confers mice with strong abilities to produce nitric oxide (NO) and cytokines. Because the peritoneal macrophages taken from the mice immunized with live or heat-killed BCG can produce NO without any accessory cells and stimulants, it is difficult to clarify the immune regulation on NO production by manipulating the macrophages. Therefore, we investigated the participation of immune T cells and cytokines in NO production by using in vitro co-cultures of macrophages from non-immune mice with T cells prepared from BCG-infected mice in the presence or absence of a mycobacterial antigen, purified protein derivative (PPD). Although the non-immune thioglycollate (TGB)-elicited macrophages could not produce any detectable NO in the presence of PPD, supplementation of the macrophage cultures with CD4+ T cells prepared from BCG-infected mice enabled the macrophages to produce NO. Immunocytostaining showed that the macrophages, hut not the immune T cells, expressed inducible NO synthase (iNOS), indicating that they were NO producers. PPD could only induce NO production if there was cell-cell contact of the CD4+ T cells in the immune cells and antigen-presenting macrophages were required for the NO production in response to PPD; this interaction led to the production of soluble mediators that induced NO production by the TGB macrophages. NO production by the co-cultured cells was abrogated by adding either anti-interferon-gamma(IFN-gamma) or anti-tumor necrosis factor alpha (TNF-alpha) antibody. Furthermore, the roles of immune T cells and PPD could be replaced by adding recombinant IFN-gamma together with TNF-alpha to the macrophage cultures, but neither alone was sufficient to induce NO production by the macrophages. Our present data indicate that TNF-alpha produced by PPD-stimulated macrophages and IFN-gamma produced by cell-cell interaction of BCG-immune T cells and antigen-engulfed macrophages together activate the macrophages to produce NO.