Reagents capable of rapid and efficient release of neuroactive amino acids (L-glutamate, GABA and glycine) upon flash photolysis of thermally stable, inert precursors have been elusive. 7-Nitroindolinyl (NI)-caged and 4-methoxy-7-nitroindolinyl (MNI)-caged compounds that fulfil these criteria are evaluated here. These caged precursors are highly resistant to hydrolysis. Photolysis is fast (half time< or =0.26 ms) and the conversion achieved with a xenon flashlamp is about 15% for the NI-caged L-glutamate and about 35% for the MNI-caged L-glutamate. A procedure is described for calibration of photolysis in a microscope-based experimental apparatus. NI-caged L-glutamate itself showed no agonist or antagonist effects on AMPA and NMDA receptors in cultured neurones, and had no effect on climbing fibre activation of Purkinje neurones. A control compound with identical photochemistry that generated an inert phosphate upon photolysis was used to confirm that the intermediates and by-products of photolysis have no deleterious effects. MNI-caged L-glutamate is as stable and fast as NI-caged L-glutamate and similarly inert at glutamate receptors, but about 2.5 times more efficient. However, NI-caged GABA is an antagonist at GABA(A) receptors and NI-glycine an antagonist at glycine receptors. The results show the utility and limitations of these fast and stable caged neurotransmitters in the investigation of synaptic processes.