1. Results presented in this paper show that there is no evidence for tunichrome in intact cells of the two Ascidiidae species Ascidia ceralodes or Phallusia julinea.
2. The yellow fluorescence of ascidian blood cells cannot be unequivocally ascribed to the yellow fluorescent tunichrome and should not be used as evidence for the compound in vivo.
3. Spectrofluorometric emission peaks reported by previous workers as evidence of tunichrome in vivo were in the same positions as water Raman bands.
4. Yellow fluorescence has been observed in the blood cells of species in which tunichrome has not been found, for example Ciona intestinalis and Pyura stolonifera.
5. The accumulated data suggest that both the lysis of morula [low in vanadium (V) and high in tunichrome precursor contents] and compartment-signet ring (high in V content) cells are necessary for the formation of tuniehrome.