The glycolipid content of human non-seminomatous germ cell tumour cell lines correlates with their differentiation lineage. To analyse whether this reflects the situation in primary tumours, we studied five embryonal carcinomas, five yolk sac tumours and nine (mixed) non-seminomas, using thin-layer chromatography and carbohydrate immunostaining. We also analysed the glycolipid content of 19 seminomas to reveal their relationship with non-seminomas. Lactosylceramide (CDH) was detected in all embryonal carcinomas, but in fewer than half of the seminomas. Seminomas and embryonal carcinomas contained globo-series glycolipids, including globotriosylceramide (Gb3), globoside (Gb4), galactosyl globoside (Gb5) and sialyl galactosyl globoside (GL7). The lacto-series glycolipid Le(X) was found in all embryonal carcinomas, but only in one seminoma. Gangliosides GD3 and GT3 were detected in many seminomas, but rarely in embryonal carcinomas. Yolk sac tumours displayed a heterogeneous glycolipid profile. Compared with seminomas and pure embryonal carcinomas, differentiated non-seminomas had reduced levels of globe-series glycolipids, especially Gb3 and Gb5, whereas CDH, Le(X), GD3 and GT3 were found in the majority of cases. Thus, the glycolipid content of non-seminoma cell lines reflects the situation in primary tumours. Globe-series glycolipids are similarly expressed in seminomas and embryonal carcinomas. The expression of Gb3 and Gb5 is reduced in non-seminomas upon differentiation. Le(X) expression in non-seminomas, including embryonal carcinomas, allows discrimination from seminomas. Expression of gangliosides in seminomas might indicate their maturation from ganglioside-negative precursor cells. Reprogramming of these precursors would result in the formation of Le(X)-expressing embryonal carcinomas.