The formation of metacyclic trypomastigotes of Trypanosoma cruzi in cultures initiated by freshly isolated bloodstream trypomastigotes was studied using three strains of parasites (Peru, CL, and Y) and two different media (L-15 and Grace's medium). Serum (10% newborn calf serum) and various sugars were required for in vitro development of metacyclic forms. This development was best at an alkaline pH (9.0); lower pH (e.g. 5.5-6.0) favored prolonged persistence of bloodstream trypomastigotes. Medium pH remained relatively stable during the 15-17 days of culture. The culture vessel employed proved very important; large numbers of metacyclic parasites were found in microtiter well cultures but not in test tube cultures. This latter result suggests that the diffusion rates of gases may be important for metacyclic trypomastigote formation in vitro.