A total of 124 samples were collected from the intestine of broiler chickens, piglet faeces, fermented foods, soils and Chinese herbs. More than 750 strains of aerobic, spore-forming bacteria were isolated from these samples. The inhibitory activity of these spore-forming strains against Escherichia coli K88, E. coli K99, Salmonella typhimurium and Staphylococcus aureus was assessed using a disc plate diffusion assay. The six bacilli with the largest inhibition zones against the four indicator bacteria were chosen and assessed for their resistance to unfavorable conditions within simulated gut environments. The strain Bacillus subtilis MA139 showed full resistance to pH 2, 0.3% bile salts and exhibited the highest antimicrobial activity. Based on these results, B. subtilis MA139 was selected as a potential probiotic and fed to piglets at concentrations of 2.2 x 10(5), 2.2 x 10(6) or 2.2 x 10(7) CFU/g of feed during a 28-day feeding trial. A negative control consisting of the basal diet with no additives and a positive control consisting of the basal diet supplemented with 16 g/ton flavomycin were also included. Ninety piglets between 35 and 40 days old were used in the in vivo animal trials. B. subtilis MA139 enhanced daily gain (P = 0.10) and feed conversion (P = 0.03) compared with the negative control. The performance of pigs fed B. subtilis MA139 supplemented diets did not differ from that of pigs fed the antibiotic diet. There was a significant increase in Lactobacilli cell counts (P = 0.02) and a numerical decrease in E. coli counts (P = 0.05) in the faecal samples of pigs fed B. subtilis MA139 with 2.2 x 10(5) CFU/g of feed. The overall results of this study show that the use of initial co-culture with indicator pathogens, a disc plate diffusion assay and simulated gut environment tolerance tests is one of effective methods of screening Bacillus for probiotic use and that B. subtilis MA139 is a promising alternative to antibiotics for use as a feed additive in piglet diets.