Light and electron microscopic observations and biochemical analysis of the lingual cartilages from the Atlantic hagfish, Myxine glutinosa, reveal two different types of cartilage, designated types 1 and 2, respectively. The anterior and medial lingual are type 1, while the posterior lingual cartilage is type 2. Chondrocytes in type 1 cartilage are similar to those found in other vertebrate cartilages. The presence within the Golgi elements of material that resembles a component of the extracellular matrix suggests the involvement of active chondrocytes in the synthesis of the matrix. The matrix of the type 1 cartilage contains fibrils arranged to form concentric lamellae in the territorial matrix and irregularly arranged, branched fibrils in the interterritorial matrix. Biochemical analysis of the type 1 cartilage reveals that it is composed primarily of a cyanogen bromide (CNBr)-insoluble protein of unique composition that we have termed “myxinin.” Myxinin appears to be similar, but not identical, to lamprin. Type 2 cartilage bears no resemblance to any other known vertebrate cartilage. The principal cells are hypertrophied and are characterized by masses of cytoplasmic filaments. The appearance of the organelles in smaller nest cells suggests that nest cells are active in the production of some of the matrix, which consists primarily of collagen. Microfibrils and a basal lamina-like material are also present. Biochemical analysis of the type 2 cartilage reveals that the CNBr-insoluble material is different from myxinin. Comparisons of lamprey and hagfish cartilages prompt the concept that these two agnathans probably followed long-independent evolutionary histories.