Several cellular signaling alterations have been identified in cystic fibrosis (CF) epithelium. One of these alterations is reduced SMAD3 protein expression and a corresponding reduction in SMAD3-mediated transforming growth factor-beta1 (TGF-beta1) signaling in CF epithelial cells compared with wild-type (wt) controls. The goal of this study was to identify a mechanism leading to reduced SMAD3 protein expression in CF epithelium. Based on previous work demonstrating isoprenoid-mediated regulation of CF-related alterations in signal transducer and activator of transcription-1 (Stat1) and inducible nitric oxide synthase (NOS2) expression, the hypothesis of this study is that inhibition of isoprenoid-dependent signaling will restore SMAD3 expression and signaling in a model of CF epithelium. Presented data will demonstrate that inhibition of both farnesyl and geranylgeranyl transferase activities partially restores SMAD3-mediated TGF-beta1 signaling and normalizes SMAD3 protein expression in one cultured model of CF cells. Analysis of the human SMAD3 promoter demonstrates that isoprenoid regulation of SMAD3 expression is dependent on Sp1/Sp3 activity, although farnesyl-mediated pathways may be acting through a secondary mechanism as well. Isoprenoid-mediated regulation of SMAD3 expression, coupled with previous data demonstrating isoprenoid control of Stat1 and NOS2 expression, suggest that the isoprenoid/cholesterol synthesis pathway is a critical intermediate in influencing CF-related cell signaling changes.