Aims: To examine sulphur (S) deprivation in combination with the presence of methane (CH4) and changes in extracellular pH as a method to enhance in situ hydrogen (H-2) generation during fermentation in the unicellular non-diazotrophic cyanobacteria Gloeocapsa alpicola and Synechocystis PCC 6803. Methodsd and Results: The level of H-2 production, measured using a gas chromatography, was determined in S-deprived cells of G. alpicola and Synechocystis PCC 6803 during fermentation. Starvation on S enhanced the rate of H-2 production by more than fourfold in both strains. S-deprived cyanobacteria were able to maintain maximum rate of H-2 production during at least 8 h of fermentation representing the entire dark period of a day. Increased H-2 production was observed during dark anoxic incubation with a gas phase of 100% CH4 (up to four times) at lower pH of the medium (5.0-5.5). Conclusions: S-deprivation in combination with CH4, added or maybe produced by another micro-organisms, and changes in the pH of the media can be used to further increase the specific capacity of unicellular non-N-2-fixing cyanobacteria to produce H-2 during fermentation with the overall aim of applying it for outdoor photobiological H-2 production. Significance and Impact of the Study: S-deprivation with respect to H-2 production is well studied in the green algae Chlamydomonas reinhardtii while its application for H-2 production in cyanobacteria is novel. Similarly, the stimulation of H-2 generation in the presence of CH4 opens up new possibilities to increase the H-2 production. Natural gas enriched with H-2 seems to be a perspective fuel and may be an intermediate step on the pathway to the exploitation of pure biohydrogen.