The ookinete is the motile form of the malaria parasite that invades the mosquito midgut epithelium to initiate sporogony. Differentiation of ingested gametocytes into ookinetes in the mosquito midgut lumen and the subsequent interaction with the luminal surface of the midgut epithelium in preparation for invasion are complex processes. To facilitate the study of these events in detail, it is necessary to produce sufficient numbers of pure, fully mature ookinetes. However, production of even a small number of Plasmodium falciparum ookinetes in vitro has proven to be a daunting task. Consequently, over the past four decades our collective understanding of the biology of this parasite form remains sorely deficient. Here, we describe a new culture technique, which improves the in vitro transformation efficiency of P. falciparum gametocytes into mature ookinetes and supports the complete development of ookinetes that retain the ability to infect the mosquito midgut and to produce oocysts.