Cell-free prepns. from ferrous ion- and sulfur-grown T. ferrooxidans prepd. under neutral (pH 7.5) or acidic conditions (pH 2.0) were compared. Under neutral conditions, the ferrous ion-oxidizing system of T. ferrooxidans was membrane-bound. At acidic conditions, the enzyme system became partially solubilized. In ferrous ion-oxidizing membranes of ferrous ion-grown cells (neutral conditions) a1-, c- and b-type cytochromes were present. The acidic prepns. contained only cytochrome a1 and c. At least 3 acid-stable c-type cytochromes (Mr 60,000, 30,000 and 25,000), an acid-stable protein with non-covalently bound heme group (Mr 20,000), and an acid-stable protein (Mr 16,000), probably rusticyanin, were detected in ferrous ion oxidizing prepns. Membranes of sulfur-grown cells prepd. under neutral conditions still oxidized ferrous ions, and contained a1-, b-, c- and in addn. an aa3-type cytochrome. Cytochrome b and aa3 were acid-labile.