Uptake of molecular hydrogen (H2) by soil is a biological reaction responsible for ∼80% of the global loss of atmospheric H2. Indirect evidence obtained over the last decades suggests that free soil hydrogenases with an unusually high affinity for H2 are carrying out the reaction. This assumption has recently been challenged by the isolation of Streptomyces sp. PCB7, displaying the high-affinity H2 uptake activity previously attributed to free soil enzymes. While this finding suggests that actinobacteria could be responsible for atmospheric H2 soil uptake, the ecological importance of H2-oxidizing streptomycetes remains to be investigated. Here, we show that high-affinity H2 uptake activity is widespread among the streptomycetes. Among 14 streptomycetes strains isolated from temperate forest and agricultural soils, six exhibited a high-affinity H2 uptake activity. The gene encoding the large subunit of a putative high-affinity [NiFe]-hydrogenase (hydB-like gene sequence) was detected exclusively in the isolates exhibiting high-affinity H2 uptake. Catalysed reporter deposition-fluorescence in situ hybridization (CARD-FISH) experiments targeting hydB-like gene transcripts and H2 uptake assays performed with strain PCB7 suggested that streptomycetes spores catalysed the H2 uptake activity. Expression of the activity in term of biomass revealed that 106–107 H2-oxidizing bacteria per gram of soil should be sufficient to explain in situ H2 uptake by soil. We propose that specialized H2-oxidizing actinobacteria are responsible for the most important sink term in the atmospheric H2 budget.