Acid- and bile-resistant Bifidobacterium strains were isolated from human feces and identified by genus-specific PCR and randomly amplified polymorphic DNA PCR. Twenty-four different strains were screened for possible production of proteinaceous antimicrobial compounds by assaying the inhibitory effects of their neutralized culture supernatants. Six Bifidobacterium strains (BIR-0304, BIR-0307, BIR-0312, BIR-0324, BIR-0326, and BIR-0349) were selected on the basis of their broad inhibitory spectra. These strains were active against gram-positive and gram-negative bacteria and yeasts relevant to food safety and human health. The antagonistic effects of the six selected Bifidobacterium strains were related to bacteriocin-like compounds, which were active at pH values between 3 and 10, stable at 100 degrees C for 10 min, resistant to alpha-amylase and lipase A, but sensitive to proteinases (trypsin, proteinase K, protease A, pepsin, and cathepsin B). The molecular masses of the antimicrobial compounds produced by Bifidobacterium BIR-0312 and BIR-0324 were in the range of 10 to 30 kDa, and those of the compounds produced by Bifidobacterium BIR-0304, BIR-0307, BIR-0326, and BIR-0349 were less than 10 kDa. All Bifidobacterium strains produced maximum antimicrobial activities in the late logarithmic phase of growth and in the presence of Tween 80. These results confirm that the synthesis of bacteriocin-like inhibitory compounds is a key factor in the in vitro inhibition of pathogen and spoilage bacteria by Bifidobacterium strains.