The cellular source(s) and mechanisms of generation of reactive oxygen species (RES) in nonphagocytic cells stimulated by cytokines are unclear. In this study, we demonstrate that transforming growth factor β1 (TGF-β1, 1 ng/ml) induces the release of H2O2 from human lung fibroblasts within 8 h following exposure to this cytokine. Elevation in H2O2 release peaked at 16 h (~ 22 pmol/min/106 cells) and gradually declined to undetectable levels at 48 h after TGF-β1 treatment. NADH consumption by these cells was stimulated by TGF-β1 while that of NADPH remained unchanged. NADH oxidase activity as measured by diphenyliodonium (DPI)-inhibitable NADH consumption in TGF-β1-treated cells followed a time course similar to that of H2O2 release. DPI, an inhibitor of the NADPH oxidase complex of neutrophils and other flavoproteins, also inhibited the TGF-β1-induced H2O2 production. Inhibitors of other enzymatic systems involving flavoproteins that may be responsible for the production of H2O2 in these cells, including xanthine oxidase, nitric oxide synthase, and both mitochondrial and microsomal electron transport systems, failed to inhibit TGF-β1-induced NADH oxidation and H2O2 production. The delay (> 4 h) following TGF-β1 exposure along with the inhibition of this process by cycloheximide and actinomycin D suggest the requirement of new protein synthesis for induction of NADH oxidase activity in TGF-β1-stimulated fibroblasts.