Gametocytes of P. falciparum are established from continuously maintained cultures of asexual blood stage parasites. Gametocytes begin to form in significant numbers in blood culture only following daily dilution with fresh RBC and medium for several days. In order for gametocytes to comprise a substantial proportion of PRBC, it is necessary for asexual PBRC to reach a high density and become stressed in vitro before conversion to gametocyte production. This occurs typically 6 to 8 d after the dilution of a culture. Gametocytes then require at least a further 8 d to reach maturity. Cultures for gametocyte production are therefore maintained for a minimum of 14 d without further dilution with fresh RBC in order to obtain a large proportion of mature and infectious gametocytes. We routinely perform gametocyte feeds to mosquitoes using cultures propagated continuously for the preceding 14 to 17 d.