We examined whether growth hormone releasing hormone GHRH may promote non-REM sleep via activation of GABAergic neurons in the preoptic area. Male Sprague Dawley rats were implanted with EEG, EMG electrodes and a unilateral intracerebroventricular (icv) cannula. Groups of rats received icv injections (3 μl) with GHRH (0.1 nmol/100 g body wt) or equal volume of physiological saline at the onset of the dark period and were permitted spontaneous sleep for 90 min. Separate groups of rats were sleep deprived by gentle handling for 90 min, beginning at the time of GHRH or saline injection, at the onset of the dark period. Other groups of rats received icv octreotide (somatostatin analogue OCT) injections, icv injection of one of two doses of competitive GHRH antagonist, or icv saline injection at light onset and were then permitted 90 min spontaneous sleep-waking. Rats were killed immediately after the 90 min sleep/wake monitoring period. Brain tissue processed for immunohistochemistry for c-Fos protein and glutamic acid decarboxylase (GAD). Single c-Fos and dual Fos-GAD cell counts were determined in the median preoptic nucleus (MnPN), and in the core and the extended parts of the ventrolateral preoptic nucleus (cVLPO and exVLPO). Icv GHRH elicited a significant increase in non-REM sleep amount. Double-labeled Fos+GAD cell counts were significantly elevated after GHRH injection in the MnPN and VLPO in both undisturbed and sleep deprived groups. OCT and GHRH antagonist significantly decreased non-REM sleep amount compared to control rats. OCT injection increased single c-Fos labeled cell counts in the MnPN, but not in the VLPO. Double-labeled cell counts were significantly reduced after OCT and the high dose of GHRH antagonist injection in all areas examined. These findings identify GABAergic neurons in the MnPN and VLPO as potential targets of the sleep regulatory actions of GHRH.