Over the past decade, several in vitro methods have been developed to study intestinal fermentation in pigs and its influence on health. In these methods, samples are fermented by a bacterial inoculum diluted in a mineral buffer solution. Generally, a reducing agent such as Na(2)S or cysteine HCl generates the required anaerobic environment by release of H(2)S inducing an imbalance among bacterial species by the production of toxic metabolites. Therefore, an experiment was conducted to study the impact of reducing agent on fermentation patterns. Protein (soybean protein and/or casein) and carbohydrate (potato starch and/or cellulose) ingredients were fermented in vitro by pig intestinal bacteria from fresh feces obtained from 3 sows fed an antibiotic-free commercial diet in 3 incubation media differing in reducing agent: (i) Na(2)S, (ii) cysteine HCl, or (iii) without reducing agent. Gas fermentation kinetics were monitored over 72 h (pressure was measured every 2 min). Short-chain fatty acid (SCFA) production after 24 and 72 h were compared among ingredient and reducing agents (n = 2). Gas production was higher (P < 0.05) when fermenting carbohydrate than protein ingredients. Except for soybean protein, total SCFA production after 24 and 72 h was similar (P > 0.05) for each ingredient regardless the incubation medium. The SCFA molar ratios did not differ (P > 0.05) between Na(2)S and without reducing agent. In conclusion, saturation of incubation media with CO(2) seems sufficient to generate an anaerobic environment. So incubation media could be simplified by omitting the reducing agent without influencing the fermentation kinetics and SCFA production.