Epimastigotes of 5 Trypanosoma cruzi stocks were cultivated in liver infusion tryptose (LIT) medium at 23-35 C or cocultivated with vertebrate cells at 35 C. A temperature decrease from 26 to 23 C resulted in a stable 60% increase in population doubling time. In zymodeme I and II stocks, a temperature increase to 35 C resulted in a transient approximately 25% increase in doubling time during the first month followed by a approximately 30% decrease after 2 mo. A zymodeme III stock did not grow at 35 C. Flow cytometric analyses showed that the total DNA/cell, guanine + cytosine (G-C), and adenine + thymidine content of 2 zymodeme II stocks increased by 3-11% when cultivated in LIT at 35 C, whereas the DNA values of 2 zymodeme I stocks did not change. The increased DNA levels, due predominantly to an increased kinetoplast G-C content, returned to normal levels when the culture temperature was reduced to 26 C. The effects of cocultivation with vertebrate cells at 35 C were identical to cultivation in LIT at 35 C except that the DNA increase in a zymodeme II stock was not stable. Total DNA/cell, nuclear, and kinetoplast DNA decreased by 8-13% upon prolonged cocultivation. No change in total protein, antigen profiles, complement sensitivity, or heat shock protein gene expression was observed as a consequence of culturing the parasites above 26 C.