Experimental variables in the manometric study of iron oxidation by washed cell suspensions of the obligate chemoautotrophThiobacillus ferrooxidanshave been examined. To obtain maximum respiration rates, extremely low cell concentrations (11–15 μg nitrogen) must be used, the substrate level must be between 400 arid 800 μmoles Fe++in the form of ferrous sulfate, and physiologically young cells must be employed. With this procedure,Qo2(N) values range from 19,000 to 22,500, nearly double any previously reported results. Optimum pH and temperature for iron oxidation are 1.75 and 40 C, respectively. Water-soluble vitamins and surfactants have no effect on the rate of respiration, Two basal salts of the growth medium (9K), i.e., potassium chloride and potassium phosphate, inhibit iron oxidation if added individually; however, concurrent addition of all the basal salts stimulates respiration significantly. Addition of small amounts of ferric iron reduces the lag and stimulates iron oxidation, whereas larger quantities inhibit respiration. During the first 5 minutes of exposure of resting cells to ferrous sulfate, ferric iron production is twice the amount predicted on the basis of oxygen consumption. Subsequently, ferric iron production levels off to approximate theoretical calculations.