Glycolipids from mucosa scrapings of small intestine of neonatal and adult pigs were tested by the thin-layer chromatogram overlay assay for the binding of Escherichia coli K99. There was practically no binding to acid or non-acid glycolipids of adult pig, known to be resistant to infection with this bacterium. However, piglets, which are susceptible to infection, showed a clear binding to a doublet band in the acid glycolipid fraction. The receptor-active glycolipid was isolated and shown by mass spectrometry, NMR spectroscopy and degradation methods to be NeuGcα-3Galsz4GlcszCer (NeuGc-GM3), the two bands being due to heterogeneity of the ceramide. When tested against various reference glycolipids, NeuAc-GM3 was shown to be inactive. This ganglioside was dominating in adult pig. The apparent developmental disappearance of N-glycolyl groups in glycolipids of intestinal mucosa may have a correspondence in protein-linked sequences as well and thus explain the resistance of adult pigs to infection with E. coli K99.