OBJECTIVE To determine if orally ingested Bacillus spores used as probiotics or direct-fed microbial feed additives germinate and the vegetative cells grow in the gastrointestinal (GI) tract. RESULTS Three independent experiments were done to determine if spores of Bacillus licheniformis and Bacillus subtilis germinate and grow in the GI tract of pigs. After a 2 weeks spore-feeding period, spores were detected in all segments of the GI tract. The lowest number of spores was found in the stomach, increasing in the small intestine to approx. 55% of the dietary inclusion. When spores were withdrawn from the feed, faecal excretion of spores reflected the dietary inclusion, but decreased gradually to the background level after 1 week. By containing spores in short, sealed pieces of dialysis membrane that were orally administered to the pigs, both the number of spores and vegetative cells could be determined by flow cytometry. Spores accounted for 72% of the total counts after 4-6 h in the stomach and proximal part of the small intestine. After 24 h, spores constituted only 12% of the total counts in the stomach, caecum, and mid-colon. Less spores and more vegetative cells were detected after 24 h, but total counts increased only 2.14-fold compared to time zero. CONCLUSIONS The experiments showed that 70-90% of dietary-supplemented Bacillus spores germinate in the proximal part of the pig GI tract, and that only limited outgrowth of the vegetative cell population occurs. The two Bacillus strains can temporarily remain in the GI system, but will be unable to permanently colonize the GI tract. CONCLUSIONS A substantial population of growing vegetative cells in the GI tract is not a prerequisite for the mode of action of Bacillus feed additives and probiotics.