A stage-specific protein has been identified in gametocytes of Plasmodium falciparum. The protein is represented on two-dimensional electrophoresis by peptides of two apparent Mr of 27,000 and 25,000, each of which has at least four different isoelectric points between pH 6.0 and 5.0. The protein is designated Pfg 27/25 (P. falciparum gametocyte-specific antigen of 27 and 25 kDa). By indirect immunofluorescence with a monoclonal antibody 1H12 specific for Pfg 27/25, this protein is present in gametocytes within 30 to 40 hr after invasion of a red blood cell by a merozoite and is present throughout subsequent maturation of the gametocyte; Pfg 27/25 is not detectable on the surface of extracellular gametes by immunofluorescence with Mab 1H12. Pfg 27/25 is absent from asexual stages of P. falciparum at any stage in their development. Pfg 27/25 is an abundant protein in gametocytes and represents between 5 and 10% of total protein of these stages. Pfg 27/25 is also a major immunogen in man during P. falciparum infection. Antibodies to this protein were readily detected in human sera from an area of holoendemic P. falciparum and also from an individual following a primary attack of P. falciparum.