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This study investigated the effect of moonlight on the nocturnal activity patterns of prairie rattlesnakes (Crotalus viridis viridis). The effect of simulated moonlight on six adults and eight juvenile prairie rattlesnakes was tested under laboratory conditions in which temperature, feeding frequency, and photoperiod were controlled. The snakes were maintained and tested under a summer photoperiod of 14L:10D h cycle. The activity of each snake was measured using an index of tracking in the sand floor of a test chamber under simulated new, half, three-quarters, and full moon light (0.06, 0.35, 1.00, and 2.10 lux, respectively). Adult snake activity was significantly greater in new moonlight (starlight only) when compared to activity in three-quarter and full moonlight. The adults also significantly increased activity in open areas in dim moonlight. Variations in simulated moonlight had no effect on the activity of the juvenile rattlesnakes or in their use of edge and open areas. Bright moonlight avoidance by adult snakes may be a strategy that reduces detection by visually hunting predators and may also be influenced by the activity patterns of their nocturnal rodent prey. The lack of moonlight avoidance by juvenile rattlesnakes may be related to utilization of nocturnally inactive prey, such as lizards and neonatal rodents.