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It has been demonstrated that rattlesnakes can discriminate between envenomed and nonenvenomed rodent prey based on venom-related cues deposited during the strike. This behavior is crucial to the snake’s ability to choose the chemical trail left by an envenomed rodent fleeing the strike area and aids in the snake’s ability to relocate the rodent. The purpose of the current study was to assess the importance of the vomeronasal system (VNS) in mediating a rattlesnake’s ability to distinguish between envenomed and nonenvenomed prey items. The VNS in adult rattlesnakes was temporarily blocked with Xylocaine ointment, and the rattlesnakes were presented with two prey items, one envenomated and one manually killed by the experimenter and not envenomed. Rattlesnakes in the Xylocaine condition failed to discriminate between envenomed and nonenvenomed prey items, whereas this discrimination was evident in the control condition. These data provide direct evidence that the VNS plays a critical role in mediating poststrike prey discrimination in rattlesnakes.