Hydrogen peroxide (H2O2) is recognized as an intra- and intercellular signalling molecule that can influence processes from embryonic development to cell death. Most research on H2O2 signalling has focused on relatively slow signalling, on the order of minutes to days, via second messenger cascades. However, H2O2 can also mediate subsecond signalling via ion channel activation. We have examined rapid signalling by H2O2 in the nigrostriatal dopamine (DA) system, which includes the substantia nigra pars compacta (SNc) and dorsal striatum, and which plays critical roles in movement and motor learning mediated by the basal ganglia. We have also examined H2O2 regulation of GABAergic neurons of the SN pars reticulata (SNr), which are the primary output neurons of the basal ganglia. All of our work has been conducted using ex vivo brain slices prepared from adult male guinea pigs or mice, after inducing deep anaesthesia (50 mg/kg sodium pentobarbital, i.p.). Methods include fast-scan cyclic voltammetry to detect DA release, whole-cell recording to monitor neuronal activity, and fluorescence imaging to indicate H2O2 generation. In guinea pig SNc DA neurons, endogenously generated H2O2 activates ATP-sensitive K+ (KATP) channels that inhibit DA neuron firing (Avshalumov et al. 2005). In guinea pig striatum, H2O2 generated downstream from glutamatergic AMPA receptors in striatal medium spiny neurons (MSNs) acts as a diffusible messenger that inhibits axonal DA release, also via KATP channels (Avshalumov et al. 2008; Patel et al. 2011). The source of dynamically generated H2O2 is mitochondrial respiration (Bao et al. 2009); thus, H2O2 provides a novel link between activity and metabolism via KATP channels. Additional targets for dynamic regulation by H2O2 include a subclass of transient receptor potential channels, TRPM2. In contrast to the inhibitory effect of H2O2 acting via KATP channels, TRPM2 channel activation is excitatory. In guinea pig brain slices, H2O2 elevation increases the excitability of striatal MSNs and increases the firing rate of SNr GABAergic neurons via TRPM2 channel activation (Bao et al. 2005; Lee et al. 2011, 2013). Notably, however, emerging evidence indicates that dynamic regulation of DA release and neuronal activity by H2O2 differs in mouse brain. Although evoked axonal DA release is modulated by glutamate and GABA acting at AMPA and GABAA receptors in the dorsal striatum of mice, as in guinea pigs, this regulation does not appear to be H2O2 dependent, indicated by a lack of effect of catalase application in mouse striatal slices. One underlying factor may be a species difference in nigrostriatal KATP channel expression. Using subunit-selective KATP channel openers, we found that axonal DA release and DA neuron activity in guinea pig brain slices can be suppressed by activation of either SUR1-containing or SUR2-containing KATP channels (Avshalumov et al. 2005; Patel et al. 2011). In mouse striatal slices, however, only SUR1-selective diazoxide, but not SUR2-selective cromakalim leads to suppression of evoked DA release. Another species difference in H2O2-dependent modulation of neuronal activity is that H2O2 elevation in mouse midbrain slices leads to inhibition of SNr GABAergic neurons via predominant KATP channel activation, as opposed to the TRPM2-dependent excitation seen with H2O2 elevation in guinea pig SNr neurons (Lee et al. 2011). This is particularly surprising given the limited regulation of DA release by H2O2-sensitive KATP channels in mouse striatum. Differences in the functional activation of H2O2-dependent KATP and TRPM2 channels between guinea pigs and mice suggest divergent roles for this regulatory process across species. The need for neuronal regulation by a metabolic signal like H2O2 might depend on unique behavioural demands across species that require differential patterns of ion channel expression. Other factors include species differences in the generation or metabolism of H2O2, for example by the glial antioxidant network – the strength of which correlates with neuron-to-glia ratio, with higher control in guinea pig (or human) vs. mouse. Regardless of these differences, however, responsiveness to H2O2 and conservation of nigrostriatal SUR1 expression in diverse species supports the idea that KATP channels are important regulators of DA neuronal function and DA release, particularly in light of previous work showing greater sensitivity of SUR1- vs. SUR2-containing channels to H2O2 and to metabolic stress.