The binding of 10 isomeric alpha-amino-heptanoic acids, of two isomeric alpha-amino-octanoic acids, of isoleucinol and valinol, and of various alpha-hydroxy acids to isoleucyl-tRNA synthetase from Escherichia coli MRE 600 has been investigated by an ultracentrifuge method. It was found that the enzyme requires a primary amino group together with a not-too-small side chain as prerequisites for ligand recognition. Though the enzyme is absolutely specific for the L isomers, it is fairly tolerant against replacement of the carboxyl group of the natural substrate by more or less hydrophobic substituents. These findings can be explained in terms of Ogston's three-point-attachment model, if it is additionally assumed that there is no further space available in the binding region normally occupied by the alpha-hydrogen atom to accept other substituents which are as bulky as the carboxyl moiety. Similarly, the architecture of the binding region of the aliphatic side chain is discussed. Our measurements show that the free energy of binding strongly depends on the size and the structure of the remainder of the molecule. None of the isoleucine analogues employed is bound as tightly as the natural substrate itself, but there is a clear preference for side chains branched at the beta-carbon atom. The functioning of the side-chain recognition site is best understood by imaging a two-finger glove, of which one finger is tailored to a methyl and the other to an ethyl group. Both these fingers, together with the binding region for the glycine moiety and a steric barrier against a fourth substituent bulkier than hydrogen, are responsible for a high steric specificity towards the one side chain over its Cbeta epimer.