Selective inhibitors of iron and sulfide oxidation, sodium azide and N-ethylmaleimide respectively, were used to demonstrate that washed cell suspensions of Thiobacillus ferrooxidans attacked both insoluble ferrous iron and sulfide during the oxidation of chalcopyrite (CuFeS2) and pyrite (FeS2). The oxidation of the two substrates occurred simultaneously and independently but the relative rates depended on how the cells were grown. When chalcopyrite-grown cells were used to oxidize chalcopyrite, 68–74% of the oxygen uptake was the result of sulfide oxidation and 25–30% the result of iron oxidation. With pyrite, all the oxygen uptake was due to sulfide oxidation. When iron-grown cells were used to oxidize chalcopyrite, two rates resulted. During the initial rapid rate, 80–90% of the oxygen uptake was due to iron oxidation, but, during the second slower rate, the result duplicated those found with chalcopyrite-grown cells. Iron-grown cells oxidized pyrite at a constant and more rapid rate than chalcopyrite-grown cells. The faster rate was due to iron oxidation; since only 20–30% of the total oxygen uptake was due to sulfide oxidation.