The effects of tunicamycin (TM) on cell adhesion and on the biosynthesis of the membrane-associated glycoproteins in aggregation-competent cells of Dictyostelium discoideum were investigated. The cohesiveness due to the EDTA-stable contact site was completely inhibited in the presence of 1 microgram/ml of TM. In contrast, when cells were treated with high concentrations of TM, the EDTA-sensitive contact site in the early developmental stage remained functional. Scanning electron microscopy showed the surface of TM-treated cells to be smoother than that of untreated cells. The membrane proteins of the aggregation-competent cells and TM-treated cells were analyzed by polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). The results showed that several protein bands at 190 kd, 170 kd, 150 kd, 135 kd, 127 kd, 94 kd, 80 kd, 68 kd, 61 kd, and 52 kd which were observed in the untreated cells disappeared in the TM-treated cells, and protein bands at 105 kd and 70 kd were newly detected in the treated cells. Tunicamycin inhibited the incorporation of [3H]mannose or [14C]glucosamine into the trichloroacetic acid (TCA)-insoluble material of the aggregation-competent cells, while it did not affect [3H]leucine incorporation. These results suggest that an asparagine-linked carbohydrate moiety of membrane-associated glycoprotein(s) is involved in cell adhesion of aggregation-competent cells of D. discoideum.