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Macaca mulatta monkeys were immunized with the candidate transmission-blocking vaccine against Plasmodium vivax, Pvs25, combined with alum or Montanide ISA 720. Efficacy was measured by combining post-immunization sera with gametocytes obtained from infections induced in chimpanzees using membrane-feeding techniques. The results indicate that immunization of M. mulatta monkeys with Pvs25 and Montanide ISA 720 was more effective than with alum in efficacy and resulted in the maintenance of a lasting transmission-blocking immunity to P. vivax. This was evident two weeks after the second immunization, and more strongly demonstrable 62 and 152 days after the second immunization. This transmission-blocking activity was strongly reinforced by a third immunization given 181 days after the primary immunization, as measured three weeks later by indirect membrane feeding. The use of gametocytes of P. vivax derived from infections induced in chimpanzees can contribute to the selection of appropriate constructs, formulations, and immunization regimens for the development of effective transmission-blocking vaccines.