Photolytic release of neurotransmitters from caged precursors is a useful method to study synaptic processes with high temporal and spatial resolution. At present, the two most widely used classes of caged precursors for studies on glutamate receptors are based on derivatives of the 2-nitrobenzyl caging group (alpha-carboxy-2-nitrobenzyl, CNB) and the nitroindoline caging group (7-nitroindoline, NI, and 4-methoxy-7-nitroindoline, MNI). Besides NI- and MNI-caged amino acids being thermally more stable than the CNB-caged amino acids, there have been no other major advantages reported of using compounds from either of these two classes. Here, we show inhibitory effects of CNB-glutamate and a number of other CNB-caged agonists on N-methyl-D-aspartate (NMDA) receptors at non-saturating concentrations of the co-agonist glycine. In contrast, NI- and MNI-glutamate and most other NI-/MNI-caged agonists that we tested were inert under these conditions. Furthermore, we demonstrate that carboxynitroindoline-caged glycine (CNI-glycine), which was previously found to inhibit glycine receptors, has no such effect on NMDA receptors. Together, these findings underline the usefulness of NI- and MNI-caged ligands and show that CNB-caged compounds should be avoided in studies involving NMDA receptors.