Milk gangliosides inhibitVibrio choleraeenterotoxin andEscherichia coliheat-labile enterotoxin. Human milk gangliosides showed considerably higher enterotoxin- inhibitory activity compared to bovine and formula milk gangliosides as measuredin vitroby enzymelinked immunosorbent assay andin vivoin rabbit small bowel loops. While gangliosides from less than 1 ml human milk inhibited 0.1 ng choleratoxinin vitroandin vivo, five to 10 times higher amounts of bovine milk gangliosides were necessary to achieve similar results. Analysis of the ganglioside composition in human, bovine, and bovine milkbased formula milk showed that the ganglioside patterns in human and bovine milk differed markedly. The ganglioside patterns of bovine milk and formula milk appeared identical. In human or bovine milk, the total amount of gangliosides was 11 mg/liter compared to 6 mg/liter in formula milk. The predominating ganglioside in human milk, monosialoganglioside 3 (74% of total gangliosides), was only a minor component (3%) of bovine milk gangliosides. Disialoganglioside 3 represented 80% of bovine milk gangliosides compared to 25% of the human milk gangliosides. Trace amounts of monosialoganglioside 1 were detected in human, as well as in bovine, milk by a sensitive high performance thin-layer chromatography immunoassay. The monosialoganglioside 1 content in human milk was 10 times higher than in bovine milk. We conclude that the higher nonimmunoglobulin enterotoxin-inhibitory activity in human milk compared to bovine milk is associated with the differences in the ganglioside fraction.