Among sulfated polysaccharides, those in the tunic of ascidians are unique: their major constituent sugar is galactose, which occurs exclusively in the L-enantiomeric form. Incorporation of D-[14C]glucose into tunic slices in vitro revealed that the cells epimerize D-glucose into L-galactose during biosynthesis of the sulfated polysaccharides. The interconversion of these two sugars involves exchange of hydrogen atoms at the epimerization sites with protons of the medium. Tunic cells also synthesize trehalose, although this disaccharide is not a prominent constituent of the tissue. Pulse-chase experiments using D-[14C]glucose reveal that incorporation of label into trehalose precedes the synthesis of the sulfated L-galactan. In addition, the loss of label from trehalose coincides with the appearance of label in the sulfated L-galactan. Based on these results, we speculate that trehalose in the ascidian tunic may be a precursor of the sulfated L-galactan.