The development of excitatory and inhibitory synaptic connections has been studied in postnatal neurons from the caudate and putamen maintained in tissue culture. Excitatory postsynaptic potentials which were sensitive to the glutamate antagonist CNQX (6-cyano-7- nitroquinoxaline-2,3-dione) appeared between 4 and 8 d in vitro. This is the first indication that glutamatergic excitatory neurons may be intrinsic to the striatum. Spontaneous inhibitory postsynaptic currents appeared at approximately the same time, several days after process outgrowth. Antibodies to glutamic acid decarboxylase (GAD), the synthetic enzyme for GABA, labeled neurons which produce bicuculline- sensitive, inhibitory postsynaptic currents. GAD immunoreactivity and immunoreactivity to synapsin I, a synaptic vesicle-associated protein, became localized to discrete sites along neurites 4–8 d after plating. It is concluded that the punctate GAD immunoreactivity identified possible sites of presynaptic transmitter release.