Acute cold exposure-induced activation of gastric myenteric neurons in conscious rats was examined on longitudinal muscle-myenteric plexus whole mount preparations. Few Fos-immunoreactive (IR) cells (<1/ganglion) were observed in 24-h fasted rats semirestrained at room temperature. Cold exposure (4 degrees C) for 1-3 h induced a time-related increase of Fos-IR cells in corpus and antral myenteric ganglia with a maximal plateau response (17 +/- 3 and 18 +/- 3 cells/ganglion, respectively) occurring at 2 h. Gastric vagotomy partly prevented, whereas bilateral cervical vagotomy completely abolished, Fos expression in the myenteric cells induced by cold exposure (2 h). Hexamethonium (20 mg/kg) also prevented 3-h cold exposure-induced myenteric Fos expression by 76-80%, whereas atropine or bretylium had no effect. Double labeling revealed that cold (3 h)-induced Fos-IR myenteric cells were mainly neurons, including a substantial number of choline acetyltransferase-containing neurons and most NADPH-diaphorase-positive neurons. These results indicate that acute cold exposure activates cholinergic as well as nitrergic neurons in the gastric myenteric ganglia through vagal nicotinic pathways in conscious rats.