Unfertilized eggs of Plecoglossus altivelis (a kind of freshwater trout, "Ayu" in Japanese) were found to contain a relatively large pool of three sialooligosaccharides. These free oligosaccharides were accumulated in the cytoplasm up to concentrations of about 35 ng per egg, which correspond to about 107 micrograms/g. Their structures were determined by a combination of chemical (composition and methylation analysis and Smith degradation) and instrumental (fast atom bombardment-mass spectrometry, secondary ion mass spectrometry, and 400-MHz1H NMR) analyses. The structures established represent a typical type of bi-, tri-, and tetraantennary sialooligosaccharides all of which end with di-N-acetylchitobiose structure (-GlcNAc beta 1-4GlcNAc) and alpha -2,3-linked NeuAc at reducing and nonreducing termini, respectively. These data show that these free sialooligosaccharides, which were originally protein-linked components, must be released during oogenesis. Although a specific functional requirement for their release is not known, the occurrence of such free sialooligosaccharides in normal animal cells or tissues has not been previously reported.