The incorporation of radioactive precursors into the extracellular basement membrane matrix has been investigated in a purified preparation of isolated rat kidney glomeruli. Using deoxycholate extraction of isolated glomeruli which were incubated with radioactively labeled amino acids and carbohydrates, as an assay system for the measurement of incorporation of basement membrane precursors into intact deoxycholate insoluble basement membrane material we demonstrated the in vitro biosynthesis of this structure. The assay system minimized the possibility of contamination of the isolated labeled basement membrane with labeled cell membrane fragments which may occur with the standard isolation procedures involving sonication and centrifugation. A linear incorporation of labeled proline, lysine, glycine, glucosamine, and galactose into glomerular basement membrane was shown. Basement membrane synthesis was inhibited by metabolic poisons and protein synthesis inhibitors as well as by inhibitors of collagen synthesis, but not colchicine, an inhibitor of collagen secretion. The appearance of 14C-hydroxyproline in the basement membrane matrix was negligible during the first 4 hours of incubation and rose only to 1% of the total proline counts thereafter. The results are consistent with the characterization of glomerular basement membrane synthesis and deposition as a two component system comprising a rapidly synthesized and deposited noncollagenous glycoprotein component(s) and a collagenous component which is only deposited after a delay of 4 to 6 hours.