The purpose of this study was to pursue the biosynthesis of proteins of human and bovine periodontal ligaments in vitro system. The excised periodontal ligaments from human and bovine were incubated in Krebs-glucose medium containing <sup>3</sup>H-proline. After incubation the incubated periodontal ligaments were homogenized and the proteins were treated with 0.1% sodium dodecyl sulfate and β-mercaptoethanol. Separation of the protein fractions was performed with agarose gel column chromatography and SDS acrylamide gel electrophoresis. The results indicated as follow: 1. Only a small percentage of <sup>3</sup>H-proline incorporated into proteins was hydroxylated to <sup>3</sup>H-hydroxyproline. 2. The labeled proteins in periodontal ligaments showed a wide distribution of molecular weight. But only small amounts of labeled protein were found that were chatacteristics of the molecular weight of collagen. 3. In all of the combined fractions of gel filtration, the degree of hydroxylation was small.