Adenosine is a potent modulator of liver fibrosis and inflammation. Adenosine has been shown to regulate such diverse activities as chemotaxis, contraction, and matrix production in hepatic stellate cells (HSC). Ecto-5'-nucleotidase/CD73 [EC 3.1.3.5] is the rate-limiting enzyme in adenosine production. Cd73-deficient mice are resistant to experimental liver fibrosis and have impaired adenosine generation. However, cell-specific expression and regulation of CD73 within the fibrotic liver have not been defined. In particular, prior evidence demonstrating that liver myofibroblasts, the cells believed to be responsible for matrix formation in the liver, express CD73 is lacking. Thus we tested the hypothesis that HSC and portal fibroblasts (PF), cells that undergo differentiation into liver myofibroblasts, express CD73 in a regulated fashion. We found that CD73 is weakly expressed in quiescent HSC and PF but is markedly upregulated at the transcriptional level in myofibroblastic HSC and PF. We furthermore found that CD73 protein and its functional activity are strongly increased in fibrous septa in rats subjected to experimental fibrosis. To determine the mechanism for the upregulation of Cd73 gene, we cloned the rat Cd73 promoter and then used serial truncation and site-directed mutagenesis to identify key regulatory elements. We identified two consensus SP1 motifs and one SMAD binding site, each of which was necessary for Cd73 gene upregulation. In conclusion, activated HSC upregulate Cd73 gene expression, via specific SP1 and SMAD promoter elements, after myofibroblastic differentiation. The ecto-5'-nucleotidase/CD73 enzyme is a novel cellular marker of activated liver myofibroblasts in vivo and in vitro and thus represents a promising molecular target for antifibrotic therapies in liver diseases.