Previous studies identifying Fe(3+) as the main oxidizing agent in CuFeS(2) bioleaching suggests that if the precipitation of additional Fe(3+) could be prevented, the Cu extraction yields should be enhanced. In this respect, the acid-generating nature of the biologically mediated oxidation of additional S(0) to H(2)SO(4) should theoretically serve as a pH regulator as well as biomass generator. This should prevent the precipitation of Fe(3+) and attenuate the biomass reduction caused by the inhibitory effect of high Fe(3+) concentrations. To prove the former hypothesis, three thermophile strains were employed for bioleaching of chalcopyrite under various additional S(0) and Fe(3+) concentrations. The hypotheses about additional S(0) application were fully confirmed; the addition of S(0) alone enhanced the leaching rates with A. brierleyi and M. sedula in media at initial pH 2. Although higher initial leaching rates were obtained with additional Fe(3+) its role as the main leaching factor is questioned; leaching with thermophiles appears to depend on the availability of protons and ORP rather than on the prevention of Fe(3+) precipitation. Additional S(0) in media with high Fe(3+) concentrations has shown the best improvements in the case of bioleaching with A. brierleyi, whereas improvements in bioleaching with M. sedula and S. metallicus were less notable. (C) 2007 Elsevier Ltd. All rights reserved.