The disulfide bonds within a variant surface glycoprotein from Trypanosoma congolense have been determined. L-[35S]Cysteine metabolically labeled protein was digested with trypsin, and radiolabeled peptides were separated by reversed-phase high performance liquid chromatography, and putative cystine-containing peptides were subdigested with other proteases and analyzed after further purification by amino acid sequencing and mass spectrometry. All eight cysteine residues of the protein, located within the N-terminal domain, are covalently linked. The four disulfide bonds are between cysteines 16/236, 171/193, 195/206, and 286/298. This is, for the first time, the determination of disulfide bonds within a variant surface glycoprotein belonging to the B-type. As all the eight cysteines of BENat 1.3 variant surface glycoprotein are positionally conserved, the cystine pattern of this protein can be regarded as a prototype of disulfide bonding within B-type variant surface glycoproteins. Although the cysteine residues of B-type variant surface glycoproteins are located at completely different positions in the protein chain compared with A-type variant surface glycoproteins, the positions of the disulfide bonds can easily be integrated into the A-type tertiary structure. This result implies that, despite their enormous amino acid sequence variability, variant surface glycoproteins, regardless of their subtype, can fold into a similar tertiary structure.