The content and distribution of gangliosides, and total lipid content, were studied in human milk samples from different periods of lactation. We found a significant correlation (r = 0.5564; p = 0.0165) between ganglioside and total lipid contents. There was a selective change in the relative concentrations of GD3 and GM3 during lactation. The most abundant ganglioside in samples from the first three weeks of lactation was GD3, whereas after the first month, GM3 was the major ganglioside. In addition to GD3 and GM3, previously known to be present in human milk, we detected several previously unreported highly polar gangliosides.