Vaccine development for Plasmodium vivax malaria is underway. A model to assess the protective efficacy of vaccine candidates in humans is urgently needed. Given the lack of continuous P. vivax cultures, we developed a system to infect Anopheles albimanus mosquitoes using blood from P. vivax-infected patients and determined parameters for challenge of malaria-naive volunteers by mosquito bite. Absence of co-infections in parasitized blood was confirmed by tests consistent with blood bank screening. A total of 119 experiments were conducted using batches of 900-4,500 mosquitoes fed by an artificial membrane feeding method. Optimal conditions for mosquito probing and infection were determined. Presence of oocyst and sporozoites were assessed on Days 7-8 and 14-15, respectively, and conditions to choose batches of infected mosquitoes for sporozoite challenge were established. Procedures to infect volunteers took a 2-hour period including verification of inoculum dose. Anopheles albimanus mosquitoes represent a valuable resource for P. vivax sporozoite challenge of volunteers.