Cellular fibronectin that contains the alternatively spliced extra domain A (EIIIA+ cFN) is upregulated after liver injury and has been reported to promote myofibroblast differentiation of precursor cells from liver, lung, and skin. We examined the role of EIIIA+ cFN in liver fibrosis induced by thioacetamide toxicity and by bile duct ligation. Surprisingly, EIIIA -/- and wild type mice were equally susceptible to fibrosis after bile duct ligation, but not after thioacetamide. We therefore studied the effects of EIIIA+ cFN on two major myofibroblast precursor populations in the liver, hepatic stellate cells and portal fibroblasts, which we suggest are the dominant cellular mediators of hepatic versus biliary fibrosis, respectively. Using a mechanically physiologic cell culture system, we found that EIIIA+ cFN was not required for myofibroblast differentiation of either cell type, but had cell-specific effects on motility. Hepatic stellate cells cultured on cFN were more motile than cells on plasma fibronectin (pFN), which lacks EIIIA, and this increased motility was dependent on EIIIA and its specific receptor, integrin α 9 β1. Portal fibroblasts, in contrast, expressed little α9 β1 and demonstrated no motility increase on EIIIA+ cFN compared to pFN. These data demonstrate that myofibroblast populations have significant functional differences regulated by subtle changes in the matrix environment, and they suggest that variable responses to matrix proteins may drive different forms of fibrosis.