In-frame deletions were introduced into each of the eight genes of the hyc operon coding for products required for the formation of the formate hydrogenlyase (FHL) system. The deletions were transferred to the chromosome and the resulting mutants were analysed for development of formate dehydrogenase H and hydrogenase 1, 2 and 3 activity. It was found that hycA, the promoter-proximal gene, is a regulatory gene and that it codes for a product counteracting transcriptional activation by FhlA. Deletions within the hycB to hycH genes specifically affected formate dehydrogenase H activity or hydrogenase 3 activity, or both. None of the mutations affected hydrogenase 1 or 2 activity. A model is proposed for the functional interaction of the different hyc operon gene products in the formate hydrogenlyase complex, which is based on the results of the mutational analysis, on the determination of the subcellular localization of the FdhF, HycE, HycF and HycG polypeptides and on the similarity of hyc gene product sequences with those from other hydrogenase systems. HycH, the product of the most promoter-distal gene, does not seem to form part of the functional FHL complex but rather is required for the conversion of a precursor form of the large subunit of hydrogenase 3 into the mature form.