Single-unit activity and unit responses to putative neurotransmitters were recorded from suprachiasmatic nucleus (SCN) neurons in brain tissue slices from ovariectomized rats either treated or untreated with estrogen. Altogether, 204 units were studied from estrogen-treated and untreated preparations, and at the resting state, 37% of these units fired regularly, 57% fired irregularly, and 6% were silent but evokable by electrical stimulation. Most of the irregular units fired continuously (n = 100), while the rest fired intermittently (n = 12) or phasically (n = 4). Neurons with different types of firing patterns also varied significantly in resting firing rate and in responsiveness to transmitters and to estrogen treatment. The average resting firing rate decreased significantly from regular, irregular and continuous, intermittent, to silent units. Acetylcholine (ACh) and/or serotonin (5-HT) injected directly into the perfusion chamber evoked responses from more irregular (69% of 61 units) than regular units (20% of 46 units). None of the 5 silent units tested was activated by ACh or 5-HT. Responses to ACh (predominantly inhibitory) and 5-HT (predominantly excitatory) seen here in vitro were opposite to those observed in vivo with iontophoretic application, and were not reversed or abolished by the blockade of synaptic transmission. Comparisons of data between the two types of preparations showed that only the responsiveness of the irregular units to ACh and to 5-HT were significantly different: both types of responsiveness were higher in estrogen-treated than in untreated preparations. No significant difference was found in the responsiveness of regular units, or in firing patterns or firing rate. Thus, the present in vitro studies have demonstrated that SCN contains a heterogeneous population of neurons distinguishable by their electrophysiological and neuropharmacological characteristics, and that estrogen has a specific action on specific types of SCN neurons.