For discrimination between threonine and 18 other naturally occurring non-cognate amino acids by the class II aminoacyl-tRNA synthetase specific for threonine, discrimination factors (D) have been determined from Kca and Km values. The lowest values were found for Cys, Met, Val (D = 70-280), indicating that threonine is only 70-280-times more often esterified to tRNA(Thr)-C-C-A than are these non-cognate compounds at the same amino acid concentrations. The highest D values have been observed for Gly, Pro, Gln, Leu, Phe, and Lys (D = 1000-2000), for the other non-cognate amino acids D values are in the medium range 300-1000. Generally, threonyl-tRNA synthetase is less specific than the class I enzymes specific for Ile, Val, Tyr, Arg, but more specific than the only investigated class II enzyme specific for Lys. In aminoacylation of tRNA(Thr)-C-C-A(2'NH2) discrimination factors D1 are in the range 2-170. From D1 values and AMP-formation stoichiometry, pre-transfer proof-reading factors II1, were determined; post-transfer proof-reading factors II2 were determined from D values and AMP-formation stoichiometry in acylation of tRNA(Thr)-C-C-A. II1 values are in the range 1.8-33, II2 values in the range 1.4-22, thus threonyl-tRNA synthetase shows the highest post-transfer proof-reading activity of six investigated synthetases (specific for Ile, Val, Tyr, Arg, Lys). Initial discrimination factors caused by differences in Gibbs free energies of binding between threonine and non-cognate amino acids have been calculated from discrimination and proof-reading factors. Assuming a two-step binding process, two factors (I1 and I2) have been determined which can be related to hydrophobic interaction forces depending on accessible surface areas of the amino acids. The threonine side chain must be bound by hydrophobic forces and two hydrogen bonds. In contrast to proof-reading factors obtained with the synthetases specific for Ile, Val, Tyr, Arg, and Lys, proof-reading factors II1 and II2 obtained with threonyl-tRNA synthetase are also related to hydrophobic interaction of the amino acid side chains and the enzyme. Threonyl-tRNA synthetase examines side chain structures of amino acids in the four postulated recognition steps, for each step the enzyme uses special distinct structures or conformations of the binding cleft.