Geobacillus tepidamans GS5-97T is a novel Gram-positive, moderately thermophilic bacterial species that is covered by a glycosylated surface layer (S-layer) protein. The isolated and purified S-layer glycoprotein SgtA was ultrastructurally and chemically investigated and showed several novel properties. By SDS-PAGE, SgtA was separated into four distinct bands in an apparent molecular mass range of 106-166 kDa. The three high molecular mass bands gave a positive periodic acid-Schiff staining reaction, whereas the 106-kDa band was nonglycosylated. Glycosylation of SgtA was investigated by means of chemical analyses, 600-MHz nuclear magnetic resonance spectroscopy, and electrospray ionization quadrupole time-of-fight mass spectrometry. Glycopeptides obtained after Pronase digestion revealed the glycan structure [→2)-α-L-Rhap-(1→3)-α-D-Fucp-(1→] n = ∼20, with D-fucopyranose having never been identified before as a constituent of S-layer glycans. The rhamnose residue at the nonreducing end of the terminal repeating unit of the glycan chain was di-substituted. For the first time, (R)-N-acetylmuramic acid, the key component of prokaryotic peptidoglycan, was found in an α-linkage to carbon 3 of the terminal rhamnose residue, serving as capping motif of an S-layer glycan. In addition, that rhamnose was substituted at position 2 with a β-N-acetylglucosamine residue. The S-layer glycan chains were bound via the trisaccharide core →2)-α-L-Rhap-(1→3)-α-L-Rhap-(1→3)-α-L-Rhap- (1→ to carbon 3 of β-D-galactose, which was attached in O-glycosidic linkage to serine and threonine residues of SgtA of G. tepidamans GS5-97 T.