The functional role of N-methyl-D-aspartic acid (NMDA) glutamate receptors in the real-time regulation of single electrical pulse (1 p)-stimulated endogenous dopamine release was investigated in slices of rat caudate putamen using fast cyclic voltammetry at a carbon fibre electrode. In the presence of Mg2+, 20 microM NMDA had a weak effect on background signals but did not affect 1 p-stimulated dopamine release. Removal of Mg2+ increased the background and doubled 1 p-stimulated dopamine release. In the absence of Mg2+, 20 microM NMDA caused a transient "release" of dopamine and decreased the background signal. The 1 p-stimulated dopamine release was subsequently reduced. In the presence of 1 microM (+/-)-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (CPP), superfusion with 20 microM NMDA did not cause a transient "release" of dopamine, and 1 p-stimulated dopamine release was not subsequently attenuated. In the presence of 1 microM tetrodotoxin, 1 p-stimulated dopamine release was abolished, but 20 microM NMDA still caused a transient "release" of dopamine. Removal of Ca2+ from the artificial CSF abolished 1 p-stimulated dopamine release and resulted in a decline in the baseline but did not affect dopamine "release" when 20 microM NMDA was added. The dopamine release-inducing effect of 20 microM NMDA was less pronounced in sites in the caudate putamen where dopamine release increased with frequency of electrical stimulation (hot spots) than in sites where there was little frequency-dependent dopamine release (cold spots). Subsequent 1 p-stimulated dopamine release was less attenuated in cold spots than in hot spots. We conclude that in the absence of Mg2+, NMDA induces release of dopamine by acting at CPP-sensitive NMDA receptors in a Ca(2+)-independent manner. This transient release depletes dopamine from a storage site from which dopamine is released by 1 p electrical stimulation. These real-time observations of the effects of NMDA on electrical stimulus-independent and -dependent dopamine release may explain the apparently conflicting observations of the effects of NMDA on dopamine release made in previous studies. They also indicate that dopamine release and storage are heterogeneous at different sites in the rat caudate putamen.