Although the periodontal ligament (PL) contains an abundance of glycosaminoglycans (GAGs), there are only a few histochemical studies describing GAGs in the developing PL. In the present study, the relationship between the formation of principal fibers and the molecular species of GAGs in the developing PL was examined by light microscopic histochemistry. Jcl:ICR mice were killed on day 0 to day 28 after birth. Paraffin-embedded tissue sections were routinely made and stained with hematoxylin-eosin (H&E), Azan, or the sensitized high iron diamine (S-HID) procedure combined with enzyme digestions. Before tooth eruption, thin threads of collagen fibers in the PL assembled and constructed principal fibers, which projected from both the side of the alveolar bone and the root of the tooth. After tooth eruption, the principal fibers from both sides were tightly entangled. In the developing PL, the molecular species of GAGs was mainly dermatan sulfate. Moreover, the relative amount of dermatan sulfate increased together with the maturation of the principal fibers, while the principal fibers adjacent to the alveolar bone and cementum contained chondroitin sulfate. These results suggest that dermatan sulfate contributes to collagen fiber assembly in the PL and that chondroitin sulfate relates to PL adhesion to the alveolar bone and to the cementum of the root.