The environment of Plasmodium falciparum gametocytes changes when they make the transition from the vertebrate to the invertebrate host. Gametocytes of this species cultivated in vitro were used to evaluate the effect of serum, pH, pCO2 tension, bicarbonate ion, and temperature on gamete formation. Temperature was the only factor responsible for keeping P. falciparum gametocytes in the inactivated state. Mature gametocytes held at temperatures above 30 degrees C remained quiescent in 10% serum, even at low ambient pCO2 tension, alkaline pH, and in the presence of 25 mM bicarbonate ion. When the temperature of the medium was allowed to drop below 30 degrees C, gametocytes emerged from the red blood cells and microgametocytes consistently exflagellated at pH 7.4, even in the absence of bicarbonate ion. With regard to bicarbonate ion, exflagellation in P. falciparum is similar to P. berghei and different from P. gallinaceum gametocytes, which have an obligate requirement for bicarbonate ion.