Hydrogenase plays an important role in biological hydrogen production. Many researchers have paid attention to this enzyme in attempts to enhance the efficiency of biological hydrogen production. In contrast to the well-characterized Fe-hydrogenase, NiFe-hydrogenase, a heterodimeric metalloenzyme composed of two subunits, has not been examined in detail for biotechnological application. It has been reported that NiFe-hydrogenases have relatively higher tolerance to oxygen than Fe-hydrogenases and particularly, some enzymes are highly oxygen-tolerant. Because inhibition by oxygen is one of the serious problems in biological hydrogen production, the development of techniques for the effective detection of oxygen-tolerant NiFe-hydrogenase genes is important. In this study, we designed a universal degenerate primer pair based on highly conserved motifs at the N-terminus of the small subunit and C-terminus of the large subunit. We successfully demonstrated that the universal degenerate oligonucleotide-primed-polymerase chain reaction (DOP-PCR) amplified 2.9-3.0 kb NiFe-hydrogenase genes from various microorganisms. This technique was applied to detect and reveal unknown sequences from the large subunit of oxygen-tolerant NiFe-hydrogenase in Hydrogenovibrio marinus. Thus, this universal DOP-PCR procedure can be successfully employed to identify or amplify novel/known oxygen-tolerant NiFe-hydrogenase genes for engineering biological hydrogen production. (C) 2007 Elsevier Inc. All rights reserved.