Conditioned environmental stimuli are known to be important determinants of drug seeking behavior. c-Fos, the protein product of the protooncogene c-Fos, is expressed in neurons when there are drug-associated cue-induced drug-seeking behaviour. Therefore, its expression could serve as a marker of regional neuronal activation. Using an extinction/reinstatement paradigm of relapse animal model, we trained Sprague-Dawley rats to nose-poke for i.v. heroin (0.05 mg/kg/infusion) either daily for 4h or 25 infusions for 14 consecutive days. We then tested these animals for cue-evoked heroin-seeking behavior after abstinence from self-administration of heroin for 14 days. Expression of c-Fos was examined in the lateral habenula (LHb), a region important for conveying information between the limbic forebrain and midbrain. Findings showed that heroin-associated conditioned stimuli could induce robust heroin-seeking behavior that was associated with increased c-Fos immunoreactivity in the medial part of the LHb. This observation suggests the involvement of the LHb in mediating drug cue-induced heroin-seeking behavior after abstinence from self-administration of heroin.