The biosynthesis of microvascular basement membrane has been demonstrated in a purified preparation of isolated bovine retinal vessels. With the use of a detergent extraction procedure to isolate morphologically intact retinal vessel basement membrane, the incorporation of labeled amino acids and carbohydrates into the purified extracellular matrix was shown. Electron microscopy demonstrated the purity of the microvessel preparations and the basement membrane derived from them, confirming that the incorporation observed represented incorporation of radioactive precursors into ultrastructurally intact microvascular basement membrane. The incorporation of amino acids and carbohydrates into the basement membrane was sensitive to heat inactivation, respiratory poisons, and inhibitors of protein and collagen synthesis. Only a small fraction of the proline label deposited in the extracellular matrix was digested by collagenase treatment, in agreement with the small amount of labeled hydroxyproline found in the isolated basement membrane, indicating that while some synthesis of collagen is observed, the bulk of incorporation occurring in this system appears to be into noncollagenous glycoprotein components of the basement membrane. The results are in agreement with different rates of secretion and deposition of glycoprotein and collagen components into the basement membrane matrix. This is the first demonstration of basement membrane biosynthesis by the isolated retinal microvasculature and represents a promising system for the study of the metabolic basis for the marked pathology of the vessel wall occurring in diseases such as diabetes mellitus. © 1979.