Obesity is one of the leading health concerns in the United States. Studies from human and rodent models suggest that inherent differences in the function of brain motivation centers, including the nucleus accumbens (NAc), contribute to overeating and thus obesity. For example, there are basal enhancements in the excitability of NAc GABAergic medium spiny neurons (MSN) and reductions in basal expression of AMPA-type glutamate receptors in obesity-prone vs obesity-resistant rats. However, very little is known about the regulation of extracellular glutamate and GABA within the NAc of these models. Here we gave obesity-prone and obesity-resistant rats stable isotope-labeled glucose (13 C6 -glucose) and used liquid chromatography mass spectrometry (LC-MS) analysis of NAc dialysate to examine the real-time incorporation of 13 C6 -glucose into glutamate, glutamine, and GABA. This novel approach allowed us to identify differences in glucose utilization for neurotransmitter production between these selectively bred lines. We found that voluntarily ingested or gastrically infused 13 C6 -glucose rapidly enters the NAc and is incorporated into 13 C2 -glutamine, 13 C2 -glutamate, and 13 C2 -GABA in both groups within minutes. However, the magnitude of increases in NAc 13 C2 -glutamine and 13 C2 -GABA were lower in obesity-prone than in obesity-resistant rats, while basal levels of glutamate were elevated. This suggested that there may be differences in the astrocytic regulation of these analytes. Thus, we next examined NAc glutamine synthetase, GAD67, and GLT-1 protein expression. Consistent with reduced 13 C2 -glutamine and 13 C2 -GABA, NAc glutamine synthetase and GLT-1 protein expression were reduced in obesity-prone vs obesity-resistant groups. Taken together, these data show that NAc glucose utilization differs dramatically between obesity-prone and obesity-resistant rats, favoring glutamate over GABA production in obesity-prone rats and that reductions in NAc astrocytic recycling of glutamate contribute to these differences. These data are discussed in light of established differences in NAc function between these models and the role of the NAc in feeding behavior.