Interleukin-1 (IL-1), tumor necrosis factor- (TNF-), and lipopolysaccharide (LPS) were examined for their ability to regulate the activity and protein levels of inducible nitric oxide synthase (NOS II) in cultured rat colonic smooth muscle cells. Treatment with these agents resulted in a time-dependent increase in NOS II activity. After 48 h, NOS II activity, measured as L-[³H]citrulline production, was increased 24.3 ± 6.9 pmol · min¹ · mg protein¹ by 1 nM IL-1 and 3.2 ± 1.1 pmol · min¹ · mg protein¹ by 1 nM TNF-, and increased synergistically by a combination of the two (51.8 ± 7.3 pmol · min¹ · mg protein¹). Measurement of NOS II activity as nitrite production yielded similar results: IL-1, 27.2 ± 1.2; TNF-, 1.6 ± 0.1; and IL-1 + TNF-, 46.8 ± 3.2 pmol · min¹ · mg protein¹ above basal. LPS (10 µg/ml) had a small but significant effect at 48 h that was only additive with that of IL-1. The increase in NOS II activity induced by IL-1 and TNF- was inhibited 73-86% by transforming growth factor-1 (TGF-1). The NOS isoform induced by IL-1 and TNF- was identified as NOS II by Western immunoblot analysis and confirmed by its 66-97% inhibition by 100 µM S-methylisothiourea, a selective NOS II inhibitor, and its Ca²⁺-independent activity. We conclude that the cytokines IL-1 and TNF- act independently and synergistically to stimulate NOS II expression and enzymatic activity in rat colonic smooth muscle through a mechanism sensitive to inhibition by TGF-1.