Previous research indicates that males prefer competition over cooperation, and it is sometimes suggested that females show the opposite behavioral preference. In the present article, we investigate the emotions behind the preferences: Do males exhibit more positive emotions during competitive than cooperative activities, and do females show the opposite pattern? We conducted two experiments where we assessed the emotional responses of same-gender dyads (in total 130 participants, 50 female) during intrinsically motivating competitive and cooperative digital game play using facial electromyography (EMG), skin conductance, heart rate measures, and self-reported emotional experiences. We found higher positive emotional responses (as indexed by both physiological measures and self-reports) during competitive than cooperative play for males, but no differences for females. In addition, we found no differences in negative emotions, and heart rate, skin conductance, and self-reports yielded contradictory evidence for arousal. These results support the hypothesis that males not only prefer competitive over cooperative play, but they also exhibit more positive emotional responses during them. In contrast, the results suggest that the emotional experiences of females do not differ between cooperation and competition, which implies that less competitiveness does not mean more cooperativeness. Our results pertain to intrinsically motivated game play, but might be relevant also for other kinds of activities.