Gametocytogenesis is a tightly regulated process marked by differentiation through distinct morphological forms and coordinated expression of sexual stage gene products. The earliest known gene product expressed at the onset of Plasmodium falciparum gametocytogenesis is Pfs16 localized on the parasitophorous vacuole membrane (PVM). Targeted gene disruption was undertaken to disrupt expression of Pfs16 and examine its potential role during sexual development. Three independent clones were demonstrated to have the coding sequence of Ps16 gene disrupted by the targeting plasmid by homologous recombination. No full-length transcripts and PVM localized 16 kDa protein were detected. Instead, all three "16ko" clones expressed a protein of 14 kDa recognized by Pfs16 specific antibodies that was mislocalized to an unidentified double membrane compartment in the parasites. Disruption of Pfs16 gene resulted in a significant reduction in gametocyte production, although the small number of gametocytes produced appeared to be normal by molecular and phenotypic evidences. Preliminary observation also suggested impaired ability of male gametocytes to exflagellate in vitro. Pfs16 does not appear to be essential for sexual development, instead may be required for optimal production of sexual parasites. Understanding mechanisms involved in the development of sexual stages of P. falciparum may identify novel targets for drugs and vaccines effective in reducing malaria transmission.