Food intake is stimulated following administration of orexin-A into the perifornical region of the lateral hypothalamus (LH/PFA). Orexin neurons originating in the LH/PFA interact with a number of hypothalamic systems known to influence food intake, including glutamatergic neurons. Glutamatergic systems in the LH/PFA were demonstrated to initiate feeding through N-methyl-d-aspartic acid (NMDA) receptors. Male Sprague-Dawley rats fitted with brain guide cannulas to the LH/PFA were used in two experiments. In the first experiment, a combination microdialysis/microinjection probe was used to deliver artificial cerebrospinal fluid (aCSF) or 500 pmol of orexin-A into the LH/PFA. Orexin-A increased interstitial glutamate to 143 +/- 12% of baseline (P < 0.05), which remained elevated over the 120-min collection period. In the second experiment, the NMDA receptor antagonist d-2-amino-5-phosphonopentanoic acid (d-AP5; 10 nmol) was administered before orexin-A. The orexin-induced increase in food intake (from 1.1 +/- 0.4 to 3.2 +/- 0.5 g, P < 0.05) during the first hour was absent in rats receiving d-AP5 + orexin-A (1.2 +/- 0.5 g). There was no effect of d-AP5 alone on food intake. These data support glutamatergic systems in the LH/PFA mediating the feeding response to orexin-A through NMDA receptors.