With c-Fos immunoreactivity as a marker for neural activity, we examined whether environmental cues associated with ethanol injection influence the expression of tolerance to ethanol-induced c-Fos activation. Over 24 training days, male Long-Evans rats received ethanol injection (2.5 g/kg) in one environment and saline injection in a different environment. Relative to rats that received ethanol for the first time, ethanol-induced c-Fos expression in the paraventricular nucleus of the hypothalamus (PVN) and the locus coeruleus (LC) was significantly reduced in rats that had received multiple prior ethanol administrations. However, tolerance was partially reversed when ethanol was given in the saline-paired, rather than the ethanol-paired, environment. Results suggest that tolerance to ethanol, as indexed by c-Fos expression in the PVN and the LC, is mediated in part by Pavlovian conditioned responses to cues that predict ethanol administration.