The biochemical characterization of bacterial glycosyltransferases involved in the assembly of cell-wall-associated polysaccharides is often hindered by the lack of the appropriate undecaprenyl-pyrophosphate-linked acceptor substrate. In order to find a suitable synthetic substrate for the alpha 1,3-mannosyltransferase AceA from Acetobacter xylinum, phytanyl-pyrophosphate-linked cellobiose was prepared. In the presence of GDP[C-14]mannose and recombinant AceA, the phytanyl-pyrophosphate-linked cellobiose afforded a C-14-labeled trisaccharide that was sensitive to alpha-mannosidase degradation in a fashion analogous to the natural undecaprenyl-pyrophosphate-linked cellobiose substrate. These results suggest that phytanyl-pyrophosphate-linked oligosaccharides may be useful substrates for other important bacterial glycosyltransferases.