Aims: The aim of this study was to develop an approach based on a reverse transcriptase (RT)-PCR/denaturing gradient gel electrophoresis (DGGE) for the detection of the functional genes nifH and anfH in Paenibacillus durus. Methods and Results: Two sets of primers were employed to study the expression of the nitrogen fixation genes in a pure-culture system of P. durus grown in media with increasing concentrations of ammonium (NH4+), tungsten (W) or molybdenum (Mo). The results obtained indicate that the expression of nitrogenase genes from P. durus can take place in the presence of relatively high levels of fixed nitrogen. It was also observed that the addition of 20 μmol l-1 molybdenum and 2 mmol l-1 tungstate did not interfere in the mRNA levels of nifH and anfH genes. Conclusions: Our results demonstrate the presence and transcription of nifH and anfH in P. durus under a variety of growth conditions. A specific set of primers was designed for the detection of the alternative system for nitrogen fixation in P. durus. Significance and Impact of the Study: The RT-PCR/DGGE system enables the rapid gathering of incremental data about the regulation of conventional and alternative nitrogenase genes in P. durus strains. © 2008 The Authors.