Repeated separation of rat pups from their mothers has been reported to increase behavioral fearfulness and hypothalamic-pituitary-adrenal (HPA) response to stress. Recently, it was suggested that it might also alter behavioral responses to natural and drug rewards. Here, we studied whether maternal separation (MS) would alter behavioral responses to a sucrose reward. We also tested whether MS would alter behavioral responses in an open-field test using a novel method of analysis [Software for the Exploration of Exploration (SEE)]. Long-Evans rat pups were exposed to either 180 min of MS, 15 min of separation [early handling (EH)] or left undisturbed [nonhandled (NH)] from postnatal day (PND) 3 to 14. The adult male offspring were tested for sucrose solution preference using a two-bottle free-choice test, operant response for sucrose under fixed ratio and progressive ratio (PR) schedules of reinforcement and response to a novel environment (open-field test). MS had no effect on sucrose preference or operant responding for sucrose reward. In the open-field test, NH rats showed a brief decrease in locomotor response, but MS rats did not differ from the NH and EH groups in the other behavioral measures. Thus, under the conditions of the present study, MS did not appear to alter reward-related processes and also had a minimal effect on open-field behavior.