Protoplasts of Saccharomyces strain 1016 took up [ 3H]glucosamine in the presence of an energy source; mannose was chosen to minimize randomization. It accumulated in the soluble intracellular pool primarily as UDP- N-acetyl[ 3H]glucosamine along with a small amount of [ 3H]glucosamine 6-phosphate. The antibiotic tunicamycin (TM) at 10 μg/ml did not affect the levels of these metabolites or inhibit the formation of the Nacetylglucosamine polymer, chitin, but did prevent the incorporation of [ 3H]glucosamine into mannan peptides and the synthesis of invertase. In vitro incorporation of [ 14C]mannose from GDP-[ 14C]mannose into mannan in a membrane preparation was not sensitive to 100 μg of TM/ml. TM appears to inhibit an N-acetylglucosaminyl transferase essential for glycoprotein biosynthesis. Binding of [ 3H]TM reflects its association with the plasma membrane fraction. This material could be recovered in an unaltered form by extraction with chloroform/methanol. If 0.2% phosphatidyl choline or phosphatidyl serine was added simultaneously with the [ 3H]TM, the binding of [ 3H]TM was greatly reduced, and the inhibitory effects of TM on protoplasts were prevented; however, addition of phospholipid 20 min later did not eliminate the inhibition, although about 80% of the bound [ 3H]TM was removed. TM interacts with lipophilic membrane components as well as inhibiting glycoprotein synthesis.