Hydrogen-oxidising bacteria play a key ecological role in a variety of habitats including the rhizosphere and hot springs. To investigate the possibly of interspecies hydrogen exchange between cyanobacteria and hydrogen-oxidising bacteria, we developed a sensitive and reliable reverse-transcriptase qPCR assay for up-regulation of the hupS gene in the knallgas bacterium Alcaligenes hydrogenophilus DSM2625. The assay detected up-regulation of the gene at initial hydrogen concentrations as low as 0.12 μM. Expression of hupS also increased in the presence of hydrogen-producing cyanobacteria, both when Ah DSM2625 was directly added to a hydrogen-producing culture of the cyanobacteria, and when cultures were physically separated in a vessel that allowed gas exchange. Additional refinements and development of the sensitive assay will lead to a better understanding of hydrogen exchange in aerobic ecosystems and development of reporter strains to detect hydrogen-producing organisms.