Structural details of keratan sulfate (KS) glycosaminoglycan, isolated from early-metamorphosing larvae (leptocephali) of bonefish (Albula sp.), are described. Bonefish KS was analyzed by first hydrolyzing the purified compound with KS endo-beta-galactosidase (keratanase) from Pseudomonas spp., and then examining the resulting oligosaccharides with reversed-phase high-performance liquid chromatography (HPLC) and 1H and 13C nuclear magnetic resonance (NMR) spectroscopy at 400 MHz. Spectral analyses were performed by COSY and HMQC. The results showed that a single oligosaccharide was produced whose structure is consistent with that of a tetrasaccharide containing two, beta-linked, N-acetyllactosamine units. Enzymic evidence indicated that the internal galactose of the tetrasaccharide was O-sulfated at C-6, and that the reducing-end galactose was unsulfated. Spectral data for C-1 of the two galactose residues were consistent with the proposed sulfation pattern. In addition, spectral evidence confirmed that a C-6 on one of the sugars was sulfated: this sulfate was tentatively assigned to the internal galactose. Chemical studies have shown that an additional sulfate group is present, but its assignment could not be confirmed, owing to the complexity of the spectral data. The known specificities of keratanase, and the production of a single tetrasaccharide, however, require that the additional sulfate reside on C-6 of either of the two available N-acetylglucosamine (GlcNAc) moieties, and that it cannot alternate between the two. The inability of beta-N-acetylglucosaminidase from beef kidney to liberate GlcNAc from the tetrasaccharide provided preliminary support for the view that this sulfate is located on the nonreducing-end GlcNAc. We conclude that the native, high molecular weight (M(r) = 55,000) KS polymer from bonefish larvae consists of a disulfated disaccharide alternating with an unsulfated disaccharide in the adjacent N-acetyllactosamine unit, with this pattern repeating itself in a regular fashion along most, or all, of the chain. This structure could provide an explanation for the ability of bonefish KS chains to self-associate into dimers. Although the N-acetyllactosamine repeat is characteristics of KS in general, the sulfation pattern is different from that postulated for the well-characterized KS chains of lower molecular weight obtained from mammalian cornea and cartilage. An additional difference was the inability to demonstrate sialic acid in bonefish KS.