Radiolabeled tyrosine is incorporated into newly biosynthesized tunichrome of Ascidia ceratodes at a rate comparable to previous findings with phenylalanine. 3H-tyrosine incorporation indicates that formation of the catechol and pyrogallol moieties occurs via the NIH shift pathway. Radiolabel is found in many compartments other than solely tunichrome (tunic > whole body ⪢ blood plasma ⋙ tunichrome). The amount of label in the tunic, whole bodies and blood plasma declines over the 14 days following exposure to 3H-tyrosine, whereas the radiolabel in the tunichrome continues to increase. These results are consistent with two alternative hypotheses: tunichromes may be formed either by sequential hydroxylation of di- and tri-peptide precursors, or by selective cleavage of a larger DOPA- or TOPA-containing compound. In either event, hydroxylation may precede formation of tunichrome's carbon backbone.