Monoclonal antibody M6704, established against the chick neural tube, was shown to recognize a trisialosyl residue, NeuAc alpha 2-8NeuAc alpha 2-8NeuAc alpha 2-3-R of C-series gangliosides. Using this antibody, the developmental changes of C-series gangliosides in fetal rat cerebral cortex have been examined. Two dimensional thin layer chromatography (TLC) enzyme-immunostaining analysis revealed that alkali treatment resulted in a great increase in GT3 that amounted to more than 85% of the total GT3 detected. The alkalilabile form was easily degraded to form GT3 by the action of the receptor-destroying enzyme of influenza C virus, sialate O-acetylesterase, indicating that the antigen was most probably 9-O-acetyl-NeuAc containing GT3. The ganglioside was highly enriched at the 14th gestation day, gradually decreased, and was not detected in adult rat cerebral cortex.