We previously reported that acute 1-day food deprivation reinstates heroin seeking in rats. The goal of the present study was to begin identifying brain sites potentially involved in this effect. For this purpose, we measured, by immunohistochemistry, the expression of c-Fos following a test for food deprivation-induced reinstatement. Groups of rats (n=9-10 per group) were trained to lever-press for heroin (0.05-0.1mg/kg/infusion) or saline for 10 days (9 h/day); each infusion was paired with a cue light. Rats were then given 10 days of extinction during which the heroin and saline syringes were removed. Next, a test for reinstatement was conducted after exposure to 0 (baseline) or 1-day food deprivation. During training, lever pressing for heroin increased over days, while responding for saline infusions paired with the cue light decreased over time. During extinction, responding on the heroin-paired lever decreased over time, while responding on the saline-paired lever remained low. In heroin-trained rats, food deprivation induced a large increase in responding on the lever associated with drug infusions. Surprisingly, food deprivation also modestly increased responding in the saline-trained rats. Food deprivation selectively increased c-Fos immunoreactivity (IR) in the prelimbic cortex of heroin-trained, but not saline-trained, rats (n=4 per condition). Food deprivation also increased c-Fos IR in both heroin- and saline-trained rats in the basolateral amygdala and the ventrolateral bed nucleus of stria terminalis (BNST), but had no effect on c-Fos expression in the dorsolateral BNST, cingulate cortex, nucleus accumbens, and central amygdala. These results raise the possibility that the prelimbic cortex is involved in food deprivation-induced reinstatement of heroin seeking.