Reinstatement of fear is a proposed mechanism for return of fear following exposure therapy. A standard reinstatement procedure in the laboratory involves a conditional stimulus (CS) paired with an unconditional stimulus (US) during acquisition (i.e., CS+), and a CS paired without an US (CS-) during acquisition. In extinction the CS + and CS- are presented alone, then the US is presented without the CS in reinstatement, and followed by test trials of the CS. The current study examined whether reinstatement of fear can be triggered by a CS that was previously paired with the US (i.e., unextinguished CS) and reduced by extinction to the CSextinguished and CSunextinguished but not the CS- in a sample of first year psychology students (expectancy data N = 93; heart rate data N = 73). A differential aversive conditioning procedure presented within a virtual reality environment was used to examine reinstatement of the US expectancy and conditioned heart rate responses. As predicted, presentation of an unextinguished CS reinstated fear of a second previously extinguished CS. Moreover, conducting extinction with multiple stimuli attenuated this reinstatement of fear as indexed by self-report and heart rate measures. The present results suggest that therapy should include exposure to multiple stimuli to reduce the likelihood of reinstatement of fear.