OBJECTIVE 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 (H2) generation during fermentation in the unicellular non-diazotrophic cyanobacteria Gloeocapsa alpicola and Synechocystis PCC 6803. RESULTS The level of H2 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 H2 production by more than fourfold in both strains. S-deprived cyanobacteria were able to maintain maximum rate of H2 production during at least 8 h of fermentation representing the entire dark period of a day. Increased H2 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-N2-fixing cyanobacteria to produce H2 during fermentation with the overall aim of applying it for outdoor photobiological H2 production. CONCLUSIONS S-deprivation with respect to H2 production is well studied in the green algae Chlamydomonas reinhardtii while its application for H2 production in cyanobacteria is novel. Similarly, the stimulation of H2 generation in the presence of CH4 opens up new possibilities to increase the H2 production. Natural gas enriched with H2 seems to be a perspective fuel and may be an intermediate step on the pathway to the exploitation of pure biohydrogen.