Cholinergic interneurons innervate virtually all medium spiny striatal cells, but the relevance of this input in regulating the activity and afferent responsiveness of these cells remains unclear. Studies in anaesthetized animals and slice preparations have shown that iontophoretic acetylcholine (ACh) either weakly excites or inhibits striatal neurons. These differential responses may reflect cholinergic receptor heterogeneity but may be also related to the different activity states of recorded units and different afferent inputs specific in each preparation. Single-unit recording was combined with iontophoresis in awake, unrestrained rats to examine the effects of ACh and selective muscarinic (oxotremorine M or Oxo-M) and nicotinic agonists (nicotine or NIC) on dorsal and ventral striatal neurons. These effects were tested on naturally silent, spontaneously active and glutamate-stimulated units. We found that iontophoretic ACh primarily inhibited spontaneously active and glutamate-stimulated units; the direction of the ACh response, however, was dependent on the firing rate. The effects of ACh were generally mimicked by Oxo-M and, surprisingly, by NIC, which is known to excite units in most central structures, including striatal neurons in anaesthetized preparation. Given that NIC receptors are absent on striatal cells but located primarily on dopamine terminals, we assessed the effects of NIC after complete blockade of dopamine receptors induced by systemic administration of a mixture of D1 and D2 antagonists. During dopamine receptor blockade the number of NIC-induced inhibitions dramatically decreased and NIC had mainly excitatory effects on striatal neurons. Thus, our data suggest that under physiologically relevant conditions ACh acts as a state-dependent neuromodulator, and its action involves not only postsynaptic but also presynaptic cholinoreceptors located on dopamine- and glutamate-containing terminals.