The obvious motor symptoms of Parkinson s disease result from a loss of dopaminergic neurons from the substantia nigra. Embryonic stem cell-derived neural progenitor or precursor cells, adult neurons and fetal midbrain tissue have all been used to replace dying dopaminergic neurons. Transplanted cell survival is compromised by factors relating to the new environment, for example; hypoxia, mechanical trauma and excitatory amino acid toxicity. In this study we investigate, using live-cell fluorescence Ca2+ and Cl- imaging, the functional properties of catecholaminergic neurons as they mature. We also investigate whether GABA has the capacity to act as a neurotoxin early in the development of these neurons. From day 13 to day 21 of differentiation [Cl-](i) progressively dropped in tyrosine hydroxylase positive (TH+) neurons from 56.0 ( 95 confidence interval, 55.1, 56.9) mM to 6.9 (6.8, 7.1) mM. At days 13 and 15 TH+ neurons responded to GABA (30 mu M) with reductions in intracellular Cl- ([Cl-](i)); from day 21 the majority of neurons responded to GABA (30 mu M) with elevations of [Cl-](i). As [Cl-](i) reduced, the ability of GABA (30 mu M) to elevate intracellular Ca2+ ([Ca2+](i)) did also. At day 13 of differentiation a three hour exposure to GABA (30 mu M) or L-glutamate (30 mu M) increased the number of midbrain dopaminergic (TH+ and Pitx3(+)) neurons labeled with the membrane-impermeable nuclear dye TOPRO-3. By day 23 cultures were resistant to the effects of both GABA and L-glutamate. We believe that neuronal susceptibility to amino acid excitotoxicity is dependent upon neuronal maturity, and this should be considered when isolating cells for transplantation studies.