Experiments were conducted to select a natural mixed microflora seed source and investigate the effect of temperature and pH on fermentative hydrogen (H 2 ) production from cattle wastewater by sewage sludge. Sewage sludge was shown to have higher cumulative H 2 production than other inoculum collected from cow dung compost, chicken manure compost, and river sludge. Experimental results show that H 2 production from cattle wastewater was significantly affected by both pH and temperature of the culture. The maximum H 2 yield was obtained at pH 5.5. H 2 yield and the ratio of butyrate/acetate (Bu/Ac) followed a similar production trend, suggesting that butyrate formation might favor H 2 production. The optimal temperature for H 2 production from cattle wastewater was 45°C with peak values of H 2 production (368 ml), hydrogen yield of 319 ml H 2 /g chemical oxygen demand (COD) consumed, and butyrate/acetate ratio of 1.43. Presence of ethanol and propionic acid indicated decreased hydrogen production; their concentrations were also affected by pH and temperature. A modified Gompertz model adequately described H 2 production and bacterial growth.