A limited number of antibiotics can be used against Helicobacter pylori infection, and resistance jeopardizes the success of treatment. Therefore, a search for new agents is warranted. The use of probiotics to enhance gastrointestinal health has been proposed for many years, but the scientific basis of the prophylactic and therapeutic actions of probiotics has not yet been clearly delineated. Probiotic strain Bacillus subtilis 3, whose safety has previously been demonstrated, is known to have antagonistic properties against species of the family Enterobacteriaceae. In the present study, it was also found to inhibit H. pylori. The anti-H. pylori activity present in the cell-free supernatant was not related to pH or organic acid concentration. It was heat stable and protease insensitive. At least two antibiotics, detected by thin-layer chromatography (R(f) values, 0.47 and 0.85, respectively) and confirmed by high-performance liquid chromatographic analysis, were found to be responsible for this anti-H. pylori activity. All H. pylori strains tested were sensitive to both compounds. One of these compounds was identified as amicoumacin A, an antibiotic with anti-inflammatory properties. MICs for H. pylori determined in solid and liquid media ranged between 1.7 and 6.8 microg/ml and 0.75 and 2.5 microg/ml, respectively. The underestimation of MICs determined in solid medium may be due to physicochemical instability of the antibiotic under these test conditions. An additive effect between amicoumacin A and the nonamicoumacin antibiotic against H. pylori was demonstrated.