The antibiotics vancomycin and aglucovancomycin have been studied by proton magnetic resonance at 270 MHz. The study has established the presence of seven amide bonds in vancomycin, six of which are secondary (-CONH-) and one primary (CONH2). The sugars vancosamine and glucose are attached as the a and p anomers, respectively. The site of attach- ment of the nitrogen atom of aspartic acid has been determined, and a carboxyl group in the molecule is shown to be part of a biphenyl-2,3'-diyldiglycinate unit. The binding of Eu3+ and Pr3+ to the carboxylate group, nuclear Overhauser effects, and the binding of the Ac-D-Ala-D-Ala to vancomycin, while not allowing detailed sttuctural proposals, do permit some general conclusions on the shape of the vancomycin molecule and the parts of vancomycin Which may be intimately involved in binding Ac-D- Ala-D- Ala. Although the glycopeptide antibiotic vancomycin was first isolated 20 years ago,' and structural itudies have been carried out by numerous groups since that the gross structure of the antibiotic (which acts by binding to mucopeptides ter- minating in the sequence D-Ala-D-Ala6) remains unknown. However, we have recently described experiments which ap- pear to account for the whole skeleton of aglucovancomycin2 in terms of four units: N-terminal D-N-methylleucine (l), aspartic acid (2), (3), and (4).7,8 Although we have provided tentative evidence8 that appropriate interconnection of these units through amide bonds (such a's to leave one free carboxy (5) and one primary amide group (6)) would account for the structure of aglucovancomycin, firm evidence for such pro- posals has been lacking. We have therefore undertaken a 270-MHz 'H NMR study of vancomycin and aglucovan- comycin and of the interaction of the former with Ac-D-Ala- D-Ala. This study has provided evidence for (i) seveq amide bonds in vancomycin, (ii) the stereochemistry of attachment of the sugars vancosamine and glucose, (iii) the sequence of attachment of some amino acid residues, and (iv) portions of the molecule which are important in binding Ac-D-Ala-D- Ala. Discussion To ensure that the study would provide useful information, it was first necessary completely to assign the spectrum of