Infants born to mothers that smoke while pregnant have a relatively high incidence of central respiratory control abnormalities. Recent studies have shown that prenatal nicotine exposure increases GABA release and the frequency of GABAergic currents, leading to an upregulation of GABAA receptors in central neurons. Activation of GABAA receptors inhibits ventilatory activity, with intense activation causing apnea. These observations lead us to hypothesize that prenatal nicotine exposure alters GABAergic control of respiratory motor pattern in the early neonatal period. Osmotic minipumps were implanted in pregnant Sprague-Dawley rats on the fifth day of gestation, and filled with nicotine (6 mg/kg/day, 2.5uL/hr) or physiologic saline (2.5 µL/hr). Brainstem-spinal cord preparations from 1-3 day old neonates were studied under in vitro conditions. Electrical activity was recorded from the fourth cervical ventral root (C4 VR), which contains the axons of phrenic motoneurons. Bath application of GABAA receptor agonists muscimol (250 µM) or pentobarbital sodium (60 µM) to the brainstem lead to consistent, reversible and significant reductions in C4 VR burst frequency. In saline-exposed animals, frequency (bursts/min) fell from 6.8 ± 0.4 to a nadir of 2.8 ± 0.5 with muscimol, and from 6.5 ± 0.3 to a nadir of 2.9 ± 0.3 for pentobarbital; in nicotine-exposed animals, frequency fell from 6.3 ± 0.4 to 1.0 ± 0.4 with muscimol and from 6.4 ± 0.2 to 1.7 ± 0.4 with pentobarbital (P < 0.05 in all cases). The decrease in C4 VR frequency was significantly greater in nicotine-exposed compared to saline-exposed preparations with both muscimol and pentobarbital (P< 0.001 for both). There were no changes in the amplitude of C4 VR bursts under any condition. The GABAA receptor antagonist bicuculline methiodide (8 µM) did not change C4 VR frequency or amplitude in either group, although it was effective in reversing the effects of muscimol. These experiments demonstrate that prenatal nicotine exposure alters the GABAergic regulation of respiratory rhythm in a reduced preparation. The results may lead to a better understanding of the perturbed breathing pattern observed in neonates that are exposed to nicotine in utero.