The development of tool compounds for the ionotropic glutamate receptors (iGluRs) remains an important research objective, as these are essential for the study and understanding of the roles of these receptors in health and disease. Herein, we report on the pharmacological characterization of (S)-2-hydroxyhistidine (2a) and (S)-2-mercaptohistidine (2b) as mediators of glutamatergic neurotransmission. While 2a displayed negligible binding affinity or activity at all glutamate receptors and transporters investigated, 2b displayed selectivity for homomeric GluK3 with binding affinities in the low micromolar range (Ki = 6.42 ± 0.74 μM). The iGluR subtype selectivity ratio for 2b was calculated at 30-fold for GluK1/GluK3, GluA3/GluK3, and GluA4/GluK3 and >100-fold for GluK2/GluK3, GluA1/GluK3, and GluA2/GluK3. Unexpectedly, functional characterization of 2b revealed that the compound is an antagonist (Kb = 7.6 μM) at homomeric GluK3 receptors while exhibiting only weak agonist activity at GluA2 (EC50 = 3.25 ± 0.55 mM). The functional properties of 2b were explored further in electrophysiological recordings of mouse hippocampal neurons.