In a study of chemosynthesis (the fixation of CO2 by autotrophic bacteria in the dark) in Thiobacillus thiooxidans, the data obtained support the following conclusions: 1. CO2 can be fixed by "resting cells" of Thiobacillus thiooxidans; the fixation is not "growth bound." 2. The physiological condition of the cell is of considerable importance in determining CO2 fixation. 3. CO2 fixation can occur in the absence of oxidizable sulfur in "young" cells. The extent of this fixation appears to be dependent upon the pCO2. 4. CO2 fixation can also occur under anaerobic conditions and the presence of sulfur does not influence such fixation. 5. However, in the CO2 fixation by cells in the absence of sulfur, only a limited amount of CO2 can be fixed. This amount is approximately 40 µl. CO2 per 100 micrograms bacterial nitrogen. After a culture has utilized this amount of CO2 it no longer has the ability to fix CO2 but releases it during its respiration. 6. Relatively short periods of sulfur oxidation can restore the ability of cells to fix CO2 under conditions where sulfur oxidation is prevented. 7. It is possible to oxidize sulfur in the absence of CO2 and to store the energy thus formed within the cell. It is then possible to use this energy at a later time for the fixation of CO2 in the entire absence of sulfur oxidation. 8. Cultures of Thiobacillus thiooxidans respiring on sulfur utilize CO2 in a reaction which proceeds to a zero concentration of CO2 in the atmosphere. 9. CO2 may act as an oxidizing agent for sulfur. 10. Hydrogen is not utilized by the organism. 11. It is possible to selectively inhibit sulfur oxidation and CO2 fixation.