We previously demonstrated a functional systemic dipsogenic response in the near-term fetal sheep (128-130 days; 145 days = full-term) with swallowing activity stimulated in response to central and systemic hypertonic saline. Preterm fetal sheep (110-115 days) do not consistently demonstrate swallowing in response to hypertonic stimuli, and it is unclear whether this is due to immaturity of osmoreceptor mechanisms or neuronal pathways activating swallowing motor neurons. To determine whether osmoreceptive regions in the preterm fetus are activated by changes in plasma tonicity, we examined Fos expression with immunostaining in these neurons in response to an osmotic challenge. Nine preterm fetal sheep [five hypertonic saline-treated fetuses (Hyp) and four isotonic saline-treated fetuses (Iso)] were prepared with vascular and intraperitoneal catheters. Seventy-five minutes before tissue collection, hypertonic (1.5 M) or isotonic saline was infused (12 ml/kg) via an intraperitoneal catheter to fetuses. Brains were examined for patterns of neuronal activation (demonstrated by Fos protein expression). Hyp demonstrated increases in plasma osmolality (~10 mosmol/kg H₂O) and Na concentrations (5 meq/l). Increased Fos expression was detected in Hyp in the organum vasculosum of the lamina terminalis (OVLT), subfornical organ (SFO), median preoptic nucleus (MnPO), supraoptic (SON), and paraventricular nuclei (PVN) compared with Iso animals. Neuronal activation within the OVLT, SFO, and MnPO indicates intact osmoregulatory mechanisms, whereas activation of the SON and PVN suggests intact fetal neural pathways to arginine vasopressin neurons. These results suggest that preterm fetal swallowing insensitivity to osmotic stimuli may be due to immaturity of integrated motor neuron pathways.