Mucus hypersecretion is a major contributor to asthma pathology and occurs as part of a spectrum of structural changes termed airway wall remodeling. Transforming growth factor (TGF)-β is proposed to play a key role in regulating airway matrix remodeling although less is known about the specific action of TGF-β isoforms in regulating mucus production.Primary human bronchial epithelial (HBE) cells cultured at air-liquid interface were treated with exogenous TGF-β(1), TGF-β(2), and/or a Th2 cytokine, interleukin (IL)-13. Expression and production of respiratory mucins, MUC5AC and MUC5B, were analyzed by real-time PCR, agarose gel electrophoresis, and western blotting. A murine-transformed Clara cell line (mtCC1-2) transfected with a luciferase reporter driven by the Muc5ac promoter containing Smad4 site-mutated cis sequences was used to determine whether exogenous TGF-β(2) affects Muc5ac promoter function.Surprisingly, TGF-β(1) showed no measurable effect on MUC5AC or MUC5B production by HBE cells whereas TGF-β(2) caused a decrease in both MUC5AC and MUC5B mRNA and protein. Dual treatment with TGF-β(2) and IL-13 partially attenuated the increase in mucin production found with IL-13 alone. This effect was confirmed by using mtCC1-2 cells where addition of TGF-β(2) reduced the ability of IL-13/EGF to induce Muc5ac promoter expression in wild-type cells; however, this decrease was absent in mutant promoter-transfected cells.Findings suggest that normal regulation of MUC5AC and MUC5B production by HBE cells is TGF-β isoform-specific and that TGF-β(2) downregulates both MUC5AC and MUC5B. Furthermore, TGF-β(2) controls baseline and IL-13-driven Muc5ac promoter function in murine Clara cells via an endogenous Smad4 recognition motif.