Pulsed Fourier transform PMR was used to study the labilization of H+ of various L-amino acids by the enzyme .gamma.-cystathionase. In the course of the normal reaction, the enzyme labilizes the .alpha. and .beta. H+ of the substrate, L-homoserine, and promotes elimination of the .gamma. substituent. .gamma.-Cystathionase also catalyzes the exchange of the .alpha. and .beta. H+ of L-amino acids which cannot undergo elimination reactions, but are competitive inhibitors of the enzyme. Both .beta. H+ of L-.alpha.-aminobutyrate, although not stereochemically equivalent, were exchanged at equal rates, whereas selectivity was shown for 1 of the .beta. H when the C chain length was increased. The data also show that .beta.-H+ exchange cannot occur without .alpha.-H+ exchange. The rate of .alpha.-H+ exchange from amino acids containing a terminal hydroxyl group at the .beta., .gamma. or .delta. C is greater than from the corresponding unsubstituted amino acid. Exchange rates of the .alpha. H+ for the inhibitors examined vary from 1/7 that of the normal enzymatic reaction to approximately the same rate as that for the elimination reaction with homoserine. An active site with 2 areas of substrate-enzyme interaction is proposed. One site contains pyridoxal 5''-phosphate and the base or bases involved in .alpha.- and .beta.-H+ exchange; the 2nd site contains a base which normally facilitates removal of the .gamma. substituent and can interact with the .gamma. and .delta. C of the substrate molecule.