Cystic fibrosis (CF) remains a fatal progressive disease in spite of the discovery and characterization of the CFTR gene. Transforming growth factor beta (TGF‐β) has been implicated in pathophysiology of CF. Previous reports have shown the trans‐Golgi network (TGN) is hyperacdified in CF epithelial cells in culture and that this hyperacidification can be corrected with the membrane permeant weak base, chloroquine. In this study bioactive TGF‐β produced by CF and normal cells was measured using a reporter cell line with a TGF‐β responsive promoter linked to luciferase. Increased levels of TGF‐β were detected in the conditioned media from CF epithelial cells compared to their matched controls—(IB3‐1 vs. S9; pCEP‐R vs. pCEP, CuFi‐4 vs. NuLi‐1). Levels of TGF‐β were normalized with chloroquine indicating that the hyperacidification of the TGN of CF cells is responsible for the altered TGF‐β levels. Pediatr Pulmonol. 2006; 41: 771–778. © 2006 Wiley‐Liss, Inc.