Increasing evidence indicates that the production of nitric oxide (NO) by inducible NO synthase (iNOS) is tightly regulated. Transforming growth factor-beta (TGF-beta) is a family of multifunctional peptides secreted during macrophage activation, but several lines of evidence suggest that TGF-beta is selectively suppressive for macrophage NO production. We therefore reasoned that a strategy employing oligodeoxynucleotides (ODN) complementary to TGF-beta mRNA (antisense ODN) might increase NO production in interferon-gamma (IFN-gamma) treated murine peritoneal macrophages. To evaluate this concept, we tested the effects of antisense ODN targeted to TGF-beta mRNA (25-mer ODN complementary to TGF-beta mRNA sequences) by introducing them into the medium of cultured macrophages. Phosphorothioation of ODN was employed to retard their degradation. Antisense ODN had no effect on NO production by itself, whereas IFN-gamma alone had a modest effect. When antisense ODN were used in combination with IFN-gamma, there was a marked cooperative induction of NO production. These effects of antisense ODN were associated with decreased TGF-beta expression in activated macrophages. However, sense ODN had no effect. Adding anti-TGF-beta antibodies to the IFN-gamma-treated macrophages mimicked the positive effect of antisense ODN on NO production. In addition, the effects of either antisense ODN or anti-TGF-beta antibodies were blocked by adding exogenous TGF-beta in cultured macrophages. These results indicate that the generation of TGF-beta by activated macrophages provides a self-regulating mechanism by which the temporal and perhaps spatial production of NO, a reactive and potentially toxic mediator, can be finely regulated.