Confluent bovine fetal tendon fibroblasts maintained in a chemically defined medium incorporated L-[6-3H]fucose and L-[5-3H]proline in a linear manner into non-diffusible macromolecules for up to 48 hrs. Equilibrium CsCl density gradient centrifugation indicated that [3H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. The [3H]fucose-labelled glycoproteins in the culture medium were analyzed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. This technique demonstrated the presence of a number of high mol. wt. fucosylated components, the most notable of which was a glycoprotein of apparent mol. wt. 150,000. Immunological procedures allowed the tentative identification of four glycoproteins including fibronectin which was found in the cell medium and in extracts of the cell layer. Two of the glycoproteins (mol. wts. 150,000 and 270,000) released into the incubation medium were shown to be related to the microfibrillar components of elastic tissue. One or more of the newly synthesized [3H]fucose labelled molecules was shown to be immunologically related to a glycoprotein (mol. wt. 60,000) extracted from bovine Achilles tendon. These studies represent the first demonstration of the synthesis of microfibril-related and tendon glycoprotein-related macromolecules by tendon fibroblasts in culture.