ABSTRACT Recently, there has been a renewed interest in the development of transmission-blocking vaccines (TBV) against Plasmodium falciparum malaria. While several candidate TBVs have been reported, studies directly comparing them in functional assays are limited. To this end, recombinant proteins of TBV candidates Pfs25, Pfs230, and PfHAP2 were expressed in the wheat germ cell-free expression system. Outbred CD-1 mice were immunized twice with the antigens. Two weeks after the second immunization, IgG levels were measured by enzyme-linked immunosorbent assay (ELISA), and IgG functionality was assessed by the standard membrane-feeding assay (SMFA) using cultured P. falciparum NF54 gametocytes and Anopheles stephensi mosquitoes. All three recombinant proteins elicited similar levels of antigen-specific IgG judged by ELISA. When IgGs purified from pools of immune serum were tested at 0.75 mg/ml in the SMFA, all three IgGs showed 97 to 100% inhibition in oocyst intensity compared to control IgG. In two additional independent SMFA evaluations, anti-Pfs25, anti-Pfs230, and anti-PfHAP2 IgGs inhibited oocyst intensity in a dose-dependent manner. When all three data sets were analyzed, anti-Pfs25 antibody showed significantly higher inhibition than the other two antibodies ( P < 0.001 for both), while there was no significant difference between the other two ( P = 0.15). A proportion of plasma samples collected from adults living in an area of malaria endemicity in Mali recognized Pfs230 and PfHAP2. This is the first study showing that the HAP2 protein of P. falciparum can induce transmission-blocking antibody. The current study supports the possibility of using this system for a comparative study with multiple TBV candidates.