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.