Ethanol induces liver fibrosis by several means that include, among others, the direct fibrogenic action of acetaldehyde on hepatic stellate cells (HSC). However the mechanisms responsible for this effect are not well understood. In this communication we investigated signal transduction pathways triggered by acetaldehyde leading to upregulation of α2(I) collagen and fibronectin gene expression in human HSC. Run-on assays showed that acetaldehyde-enhanced transcription of these 2 genes as early as 2 hours, via de novo protein synthesis-independent and -dependent mechanisms. It also stimulated a time-dependent induction in phosphorylation of pp70S6K and extracellular-regulated kinase ½ (ERK½). These effects were completely prevented by calphostin C, a protein kinase C inhibitor. As expected, acetaldehyde-elicited ERK½ phosphorylation was inhibited by PD98059, a MEK inhibitor, but not by wortmannin, a PI3K inhibitor. On the other hand, both of these inhibitors partially inhibited phosphorylation of pp70S6K induced by acetaldehyde suggesting that its activation is ERK½- and PI3K-dependent. Acetaldehyde-elicited fibronectin and α2(I) collagen upregulation was inhibited by calphostin C. However, while PD98059, wortmannin and rapamycin (a pp70S6K inhibitor) completely abrogated α2(I) collagen upregulation, they had no effect on fibronectin expression. Overall, these data suggest that protein kinase C is an upstream component from which acetaldehyde signals are transduced to other pathways such as PI3K and ERK½. In addition, differential activation of these pathways is needed for the increase in fibronectin and α2(I) collagen gene expression induced by acetaldehyde in human HSC.