Contextual stimuli present during a single lifetime cocaine self-administration experience acquire occasion-setting actions sufficient to persistently elicit cocaine-seeking behavior in rats, with effects lasting nearly 1 year. The goal of this study was to identify neural substrates mediating the acquisition of drug-related conditioning taking place during a single cocaine self-administration experience with focus on the subicular formation, a brain site that has been implicated in associative learning relevant for conditioned reward-seeking including conditioned reinstatement. Male Wistar rats were given 2 h of response-contingent access to intravenous cocaine or saline in the presence of distinct stimuli that served as contextual stimuli associated with the availability and subjective effects of cocaine (S(+)) vs saline (S(-)). Before onset of the sessions, rats received bilateral microinjections of tetrodotoxin (TTX) into the ventral subiculum (VSUB) or dorsal subiculum (DSUB). Following extinction of responding by withholding cocaine, rats were subjected to reinstatement tests in which exposure to the cocaine- (but not saline) associated stimulus produced strong recovery of responding. This effect was completely abolished in rats with transient TTX inactivation of the DSUB during the conditioning session. TTX inactivation of the VSUB during conditioning did not alter the response-reinstating effects of the cocaine cue. The results suggest that functional integrity of the DSUB, but not VSUB, is critical for the acquisition of conditioned cocaine-seeking controlled by contextual stimuli under conditions where such learning occurs during a single conditioning trial.