Biologically active recombinant human follitropin has been expressed in Chinese hamster ovary cells. The carbohydrate chains of the recombinant glycoprotein hormone were enzymatically released by peptide-N4-(Nacetyl-p-glucosaminy1)asparagine amidase F. The oligosaccharides were separated from the N-deglycosylated protein by gel-permeation chromatography on Bio-Gel P-100, and fractionated by a combination of FPLC on Mono Q and HPLC on Lichrosorb-NH,. The structures of the carbohydrate chains were determined by 500-or 600-MHz 'H-NMR spectroscopy. The following types of carbohydrates occur: monosialylated diantennary (lo%), disialylated diantennary (43 YO), disialylated tri-antennary (5%), trisialylated tri-antennary (1 3%), trisialylated tri'-antennary (So/,), and tetrasialylated tetraantennary (1 2%) N-acetyllactosamine type of carbohydrate chains, all bearing exclusively a2-3-linked N-acetylneuraminic acid (Neu5Ac). Previously, for pituitary follitropin mono-, di-, tri-, tri'-, and tetra-antennary oligosaccharides containing a2-3-as well as a2-6-linked Neu5Ac residues were reported. The bisecting GlcNAc residues present in native follitropin were not detected in the recombinant glycoprotein. Of the oligosaccharides 29% have an al-6-linked Fuc residue at the asparagine-bound GlcNAc, whereas this amount is about 50% in pituitary follitropin. In some of the tri-, tri'-and tetra-antennary oligosaccharide fractions small amounts (< 5%) of compounds were detected having one or more additional Nacetyllactosamine units.Follitropin (follicle-stimulating hormone, FSH) belongs to a heterodimeric glycoprotein hormone family in which each hormone is composed of two non-covalently linked subunits, denoted a and / 3. The amino acid sequences of the a subunits are essentially identical, whereas those of the fl subunits are hormone-specific. The structures of the carbohydrate chains differ between the glycoprotein hormones and are believed to confer hormone specific-functions [l -31.FSH is synthesized by the gonadotropes in the anterior pituitary and is required for normal reproductive functioning in both females and males. There is a need of FSH for clinical purposes since it is applied, either alone or in combination with human LH, for controlled ovarian hyperstimulation in in vitro fertilization [4,5]. Human FSH is also used to stimulate the development of ovarian follicles in anovulatory women suffering from luteal phase deficiency [6], or chronic anovulatory syndrome [7].Correspondence to J. F. G. Vliegenthart, Bijvoet Center, Department of Bio-Organic Chemistry, Utrecht University, Transitorium 111, P. 0. Box 80.075, NL-3508 TB Utrecht, The NetherlandsAbbreviations. PNGase-F, peptide-N4-(N-acetyl-P-glucosaminy1)asparagine amidase F ; NeuSAc, N-acetylneuraminic acid; Fuc, Lfucose; hFSH, human follicle-stimulating hormone (follitropin); LH, luteinizing hormone (lutropin); hCG, human chorionic gonadotropin; CHO, Chinese hamster ovary; t-PA, tissue plasminogen activator.Enzymes. Peptide-N4-(N-acetyl-~-glucosaminyl)asparagine a...