Objective. To study donors’ motivation and ambivalence before donation of gametes. Design. Cross-sectional study. Setting. Seven Swedish university hospital clinics. Sample. Of the 220 eligible oocyte donors and 156 eligible sperm donors who were approached, 181 (82%) oocyte donors and 119 (76%) sperm donors agreed to participate. Methods. Gamete donors completed a questionnaire in the clinic prior to the donation. Main outcome measures. Motives and ambivalence towards donation. Results. In general, gamete donors donated for altruistic reasons (95%). A greater percentage of oocyte than sperm donors had a personal experience of biological children, which motivated them to donate (65 vs. 32%). A greater percentage of sperm donors compared with oocyte donors were curious about their own fertility (24 vs. 9%), and they also believed that they were contributing what they regarded as their own good genes to other couples (45 vs. 20%). Prior to donation, potential sperm donors were more ambivalent towards donating than were oocyte donors (39 and 21%, p < 0.001). Conclusions. The motives to donate gametes are mainly altruistic. We conclude that men and women differ in their view towards donating gametes. Sperm donors had a higher degree of ambivalent feelings towards donation than oocyte donors.