Structural adaptation of the blood vessel wall occurs in response to mechanical factors related to blood pressure and flow. To elucidate the relative roles of pressure, flow, and medium composition, we have developed a novel organ culture system in which rabbit thoracic aorta, held at in vivo length, can be perfused and pressurized at independently varied flow and pressure for several days. Histology and histomorphometry, as well as scanning electron microscopy, revealed a well-preserved wall structure. In arteries perfused and pressurized at 80 mm Hg, endothelial injury led to a 2-fold increase in [3H]thymidine incorporation in the media, which peaked at 3 to 5 days and returned to baseline level at 6 to 8 days. In intact endothelialized vessels cultured for 3 days under no-flow conditions, pressure per se had no effect on DNA synthesis. In contrast, in the presence of serum, total protein synthesis, as assessed by [35S]methionine incorporation into the media, was enhanced 6-fold at 150 mm Hg compared with vessels pressurized at 0 or 80 mm Hg. In intact vessels perfused at a constant flow of 40 mL/min for 3 days, DNA synthesis was unchanged regardless of the pressure level when vessels were cultured in the presence of serum but increased 8-fold at both 80 and 150 mm Hg in the absence of serum. Unlike DNA synthesis, total protein synthesis was enhanced 12-fold by flow regardless of the presence or absence of serum. Expression of fibronectin was markedly enhanced at high transmural pressure, and serum potentiated its expression in the arterial wall. This novel organ culture system of perfused and pressurized vessels allowed identification of differential effects of pressure, flow, and serum on DNA and total protein synthesis, including cellular fibronectin expression.