In the striatum, interneurons have not been as well characterized physiologically as the spiny projection cells. We found that the neostriatal interneurons can be divided at least into three classes by physiological, chemical and morphological criteria. The first was FS cells (fast-spiking cells) which fired very short-duration action potentials at constant spike frequency during depolarizing pulses, were immunoreactive for parvalbumin (calcium-binding protein), and had axons with very dense collateralization within or near their dendritic fields. Another class was identified as those which fired low-threshold spikes (LTS cells) from hyperpolarized potentials, were positive for somatostatin and nitric oxide synthase (NOS), and had the largest axonal fields. The other class of interneurons had longer-lasting afterhyperpolarizations (LA cells), were positive for choline acetyltransferase, and were mostly large aspiny cells. Glutamic acid decarboxylase (GAD67) or GABA immunoreactivity was detected at the somata or terminals of parvalbumin FS cells and somatostatin/NOS LTS cells, but not of cholinergic LA cells. Substance P, probably released from the collaterals of cells projecting to the substantia nigra, excited LA cells and LTS cells, but not FS cells. These results suggest that the striatum has at least one type of cholinergic and two types of GABAergic interneurons which are different in physiological, chemical and pharmacological characteristics.