The clinical literature suggests that exposure to environmental stimuli previously associated with heroin availability may precipitate relapse. However, experimental studies elucidating the significance of learned associations between drug availability and reinstatement of heroin-seeking behavior in the rat are still scarce. To examine the role of environmental stimuli in reinstatement of heroin-seeking behavior, rats were trained to associate discriminative stimuli (DS+) with intravenous heroin availability vs. nonreward [i.e., availability of intravenous saline (DS-)]. The animals then were subjected to extinction training during which the discriminative stimuli were not presented, and lever pressing did not result in drug or saline infusion. The resistance to extinction varied greatly among animals (2.5-11.4 weeks). When the discriminative stimuli were reintroduced, the DS+ reinstated responding while the DS- did not. The average number of responses for heroin during the reinstatement trial (12.8) paralleled the average responding for heroin during discrimination training (12.6), suggesting that the associations between environmental stimuli and drug availability are long-lasting and powerful motivators of drug-seeking behavior.