Increased extracellular matrix protein production leading to structural abnormalities is a characteristic feature of chronic diabetic complications. We previously showed that high glucose in endothelial cell culture leads to the upregulation of basement membrane protein fibronectin (FN) via an endothelin (ET)-dependent pathway involving activation of NF-kappaB and activating protein-1 (AP-1). To delineate the mechanisms of basement membrane thickening, we used an animal model of chronic diabetes and evaluated ET-dependent activation of NF-kappaB and AP-1 and subsequent upregulation of FN in three target organs of chronic diabetic complications. After 3 mo of diabetes, retina, renal cortex, and myocardium demonstrated increased FN mRNA and increased ET-1 mRNA expression. Increased FN expression was shown to be dependent on ET receptor-mediated signaling, as the increase was prevented by the dual ET receptor antagonist bosentan. NF-kappaB activation was most pronounced in the retina, followed by kidney and heart. AP-1 activation was also most pronounced in the retina but was similar in both kidney and heart. Bosentan treatment prevented NF-kappaB activation in the retina and heart and AP-1 activation in the retina and kidney. These data indicate that, although ETs are important in increased FN production due to diabetes, the mechanisms with respect to transcription factor activation may vary depending on the microenvironment of the organ.