Recently, synthesis and secretion of connective tissue growth factor (CTGF)/CYR61/CTGF/NOV‐family member 2 (CCN2) in cultures of hepatocytes were shown, which are sensitively up‐regulated by exogenous TGF‐β. In this study TGF‐β‐dependent CTGF/CCN2 expression in hepatocytes cultured under completely TGF‐β‐free conditions was analysed by Western‐blots, metabolic labelling, and CTGF‐reporter gene assays. In alkaline phosphatase monoclonal anti‐alkaline phosphatase complex (APAAP)‐staining of cultured hepatocytes it was demonstrated that latent TGF‐β within the hepatocytes becomes rapidly detectable during culture indicating an intracellular demasking of the mature TGF‐β antigen. Subsequent signaling to theCTGF/CCN2 promoter occurs via p‐Smad2, whereas p‐Smad3 does not seem to be involved. Cycloheximide did not abolish the rapid immunocytochemical appearance of mature TGF‐β, but calpain inhibitors partially suppressed intracellular TGF‐β activation and subsequently CTGF up‐regulation. Calpain treatment had the reverse effect. None of the inhibitors of extracellular TGF‐β signalling was effective in the reduction of spontaneous CTGF synthesis, but intracellularly acting Alk 4‐/Alk 5‐specific inhibitor SB‐431542 was able to diminish CTGF expression. The assumption that latent intracellular TGF‐β is activated by calpains during culture‐induced stress or injurious conditions in the liver in vivo was further validated by a direct effect of calpains on the activation of recombinant latent TGF‐β. In conclusion, these data are the first to suggest the possibility of intracrine TGF‐β signalling due to calpain‐dependent intracellular proteolytic activation leading to transcriptional activation of CTGF/CCN2 as a TGF‐β‐sensitive reporter gene. This mechanism might be deleterious for keeping long‐term hepatocyte cultures due to TGF‐β‐induced apoptosis and, further, might be of relevance for induction of apoptosis or epithelial‐mesenchymal transition of hepatocytes in injured liver.