The effect of iontophoretically applied acetylcholin (ACh) was investigated in area CA1 of transverse hippocampal slices maintained in vitro. In synaptically activated regions of the dendritic field, ACh reduced the amplitude of the population spike recorded from the pyramidal layer. In dendritic regions which were not synaptically activated, ACh increased the amplitude of the population spike or it had no effect. The depressing effect of ACh was abolished in denervated dendritic regions. The intracellularly recorded excitatory postsynaptic potential (EPSP) decreased in amplitude when ACh was applied at the synaptic site. The resting membrane potential, the time course of the EPSP, and the membrane resistance were unaffected. ACh increased the excitability of afferent fibers and this was independent of synaptic transmission. We conclude that ACh in addition to its postsynaptic effects has a presynaptic site of action.