Aflatoxins are mycotoxins that cause health and economic problems when they contaminate food and feed. One potential method for reducing human health effects due to aflatoxin ingestion is to block uptake via binding by bacteria that either make up the normal gut flora or are present in fermented foods in our diet. These bacteria would bind aflatoxin and make it unavailable for absorption in the intestinal tract. Bifidobacteria comprise a large fraction of the normal gut flora, are thought to provide many probiotic effects and are increasingly used in fermented dairy products. These qualities targeted bifidobacteria for studies to determine if various strains of heat-killed bifidobacteria can bind aflatoxin B1 (AFB1) in vitro. The AFB1 binding affinities of various strains of bifidobacteria, Staphylococcus aureus, and Escherichia coli were quantitated utilizing enzyme-linked immunosorbent and [3H]AFB1 binding assays. The bacteria analyzed were found to bind significant quantities of AFB1 ranging from 25% to nearly 60% of the added toxin. The data also suggest that there are reproducible strain differences in AFB1 binding capacity.