ABSTRACT Microbes can produce molecular hydrogen (H ₂ ) via fermentation, dinitrogen fixation, or direct photolysis, yet the H ₂ dynamics in cyanobacterial communities has only been explored in a few natural systems and mostly in the laboratory. In this study, we investigated the diel in situ H ₂ dynamics in a hot spring microbial mat, where various ecotypes of unicellular cyanobacteria ( Synechococcus sp.) are the only oxygenic phototrophs. In the evening, H ₂ accumulated rapidly after the onset of darkness, reaching peak values of up to 30 μmol H ₂ liter ⁻¹ at about 1-mm depth below the mat surface, slowly decreasing to about 11 μmol H ₂ liter ⁻¹ just before sunrise. Another pulse of H ₂ production, reaching a peak concentration of 46 μmol H ₂ liter ⁻¹ , was found in the early morning under dim light conditions too low to induce accumulation of O ₂ in the mat. The light stimulation of H ₂ accumulation indicated that nitrogenase activity was an important source of H ₂ during the morning. This is in accordance with earlier findings of a distinct early morning peak in N ₂ fixation and expression of Synechococcus nitrogenase genes in mat samples from the same location. Fermentation might have contributed to the formation of H ₂ during the night, where accumulation of other fermentation products lowered the pH in the mat to less than pH 6 compared to a spring source pH of 8.3. IMPORTANCE Hydrogen is a key intermediate in anaerobic metabolism, and with the development of a sulfide-insensitive microsensor for H ₂ , it is now possible to study the microdistribution of H ₂ in stratified microbial communities such as the photosynthetic microbial mat investigated here. The ability to measure H ₂ profiles within the mat compared to previous measurements of H ₂ emission gives much more detailed information about the sources and sinks of H ₂ in such communities, and it was demonstrated that the high rates of H ₂ formation in the early morning when the mat was exposed to low light intensities might be explained by nitrogen fixation, where H ₂ is formed as a by-product.