Aims: To study Bacillus clausii from a pharmaceutical product (Enterogermina O/C, N/R, SIN, T) and reference strains (B. clausii and Bacillus subtilis) for eco-physiological aspects regarding the gut environment. Methods and Results: Spores and vegetative cells were challenged in vitro miming the injury of gastrointestinal transit: pH variations, exposure to conjugated and free bile salts, microaerophilic and anaerobic growth. No relevant differences were found studying the growth at pH 8 and 10, whereas at pH 7 the yields obtained for O/C and SIN were higher than those obtained for N/R and T strains. The spores were able to germinate and grow in the presence of conjugated bile salts (up to 1%, w/v) or free bile salts (0(.)2%) and also exhibited tolerance for the combined acid-bile challenge. As evidenced by lag-time, growth rate and cell yield the tolerance of Enterogermina isolates for conjugated salts was comparable with that of B. clausii type strain (DSM 8716(T)), and resulted higher than that observed for B. subtilis (ATCC 6051(T)). All the considered B. clausii strains demonstrated microaerophilic growth, but only some grew anaerobically in a nitrate medium. Conclusions: The ability of B. clausii spores to germinate after an acid challenge and grow as vegetative cells both in the presence of bile and under limited oxygen availability is consistent with the beneficial health effects evidenced for spore-forming probiotics in recent clinical studies. Significance and Impact of the Study: The experimental evidence from this study emphasizes some functional properties of B. clausii strains regarding their use as probiotics.