Lung fibrosis is a fatal condition of excess extracellular matrix (ECM) deposition associated with increased transforming growth factor β (TGF‐β) activity. Although much is known about its pathological features, our understanding of the signal transduction pathways resulting in increased ECM and collagen deposition in response to TGF‐β is still incompletely defined. We have previously reported that a JunD homodimer of the transcription factor AP‐1 is specifically activated by TGF‐β in lung fibroblasts. Here we demonstrate that JunD is also specifically required for TGF‐β‐induced effects. Antisense against JunD, but not c‐fos or c‐jun, significantly inhibited collagen deposition in response to TGF‐β in primary human lung fibroblasts. We then investigated the ability of pharmacological agents to inhibit TGF‐β‐induced signaling and collagen deposition. Cs‐A and IFN‐γ, but not glucocorticoids, cyclophosphamide, or azathioprine, inhibited TGF‐β‐induced signaling, as assessed by luciferase reporter gene assays, and collagen deposition. TGF‐β antagonism by Cs‐A was associated with direct inhibition of JunD activation, as demonstrated by electrophoretic mobility shift analyses. In contrast, the effects of IFN‐γ required signal transducer and activator of transcription (STAT)‐1. We thus identify the JunD isoform of AP‐1 as an essential mediator of TGF‐β‐induced effects in lung fibroblasts. TGF‐β‐induced signaling and collagen deposition are efficiently antagonized by Cs‐A and IFN‐γ treatment, both of which exhibit distinct molecular mechanisms of action. These observations therefore offer novel targets for future therapy of fibrotic lung disease.—Eickelberg, O., Pan‐sky, A., Koehler, E., Bihl, M., Tamm, M., Hildebrand, P., Perruchoud, A. P., Kashgarian, M., Roth, M. Molecular mechanisms of TGF‐β antagonism by interferon γ and cyclosporine A in lung fibroblasts. FASEB J. 15, 797‐806 (2001)