With DNA sequence information available for Thiobacillus ferrooxidans it will become much easier to understand its physiology and genetics. However, based on DNA sequence information alone a number of its genes, especially those involved in the more unusual or specific functions of the microorganism such as iron oxidation, could remain without a clear assignment of function. We show how naturally occurring insertion sequences can be used to generate mutants by knocking out gene function and how these knock-out mutants can be analyzed to reveal the gene involved in the mutation. An iron oxidizing mutant of T. ferrooxidans ATCC19859 is shown to contain a copy of the insertion sequence IST1 in a gene, which we term resB, that is probably involved in the biogenesis of a c-type cytochrome. Downstream of resB, and perhaps part of the same transcriptional unit, is another gene, resC, also probably involved in the biogenesis of a c-type cytochrome. Some of the predicted properties of these biogenesis proteins and their evolutionary relations are described. The target cytochrome(s) of the putative cytochrome-c type biogenesis proteins remains unknown, although just upstream of resB is a gene potentially encoding a cytochrome cl, making it a likely candidate as the target cytochrome. The target cytochrome is essential for growth on iron but not for growth on thiosulfate and may prove specific for the pathway of energy transduction and/or electron flow during iron oxidation.