A monoclonal antibody (MAb) that reacted with the cell-surface antigens of adrenocortical cells was generated against cell suspensions from guinea pig adrenal glands. Cell-surface membranes of the adrenocortical cells in all zones, i.e., zona glomerulosa, zona fasciculata, and zona reticularis, were labeled with the antibody. Adrenal medulla remained unlabeled. Immunoelectron microscopy showed that entire plasma membranes, i.e., plasma membranes between adjacent cells and free cell-surface membranes, including sinusoidal microvilli, were immunoreactive to the antibody. Immunoblot analysis demonstrated that the antibody bound to two prominent bands at molecular weights of approximately 62,000 and 110,000. Two bands were stained with lectin-digoxigenin conjugates. The 110 KD band reacted with Datura stramonium (DSA) and Maackia amurensis (MAA) agglutinins, indicating the presence of N-acetyl-glucosamine and sialic acid-linked alpha (2-3) to galactose; the 62 KD band reacted with SNA, indicating the presence of sialic acid-linked alpha (2-6) to galactose. In adrenocortical cells, the reaction pattern of Sambucus nigra (SNA) agglutinin was similar to that of the (MAb), whereas reaction patterns of DSA and MAA were different. Both neuraminidase digestion and prior absorption of the antibody with N-acetyl-neuraminic acid completely prevented the immunolabeling of adrenocortical cells. These results indicate that the MAb mainly recognizes the 2-6 sialylated cell-surface antigen of adrenocortical cells.