Ten microM glycine, D-serine and D-alanine potentiated L-glutamate (30 microM)-induced contractions of the guinea pig ileum by an average of 35, 53 and 24%, respectively. On the contrary, D-cysteine, at the same concentration, caused a 21% inhibition of the contractile response to L-glutamate. This inhibitory effect of D-cysteine was abolished by 10 microM glycine. The corresponding L-isomers of these amino acids, namely L-serine, L-alanine and L-cysteine and the other amino acids tested, possessed negligible activity or were inactive in this test. The IC50 values of the same compounds for strychnine-insensitive binding of [3H]glycine (20 nM) to cortical membranes from the brain of the rat were: 0.26 microM, glycine; 1.2 microM, D-serine; 2.1 microM, D-alanine; 8.6 microM, D-cysteine; 51 microM, L-serine; 90 microM, L-alanine; greater than 1000 microM, L-cysteine. On the whole, these results point out a strict requirement for stereoselectivity for both of the effects examined. In addition, the results obtained in the ileum preparation suggest that D-cysteine may act as an antagonist, rather than as an agonist at the glycine site which regulates the responses of N-methyl-D-aspartate receptors.