Enlargeosomes are cytoplasmic organelles discharged by regulated exocytosis, identified by immunofluorescence of their membrane marker, desmoyokin/Ahnak, but never revealed at the ultrastructural level. Among the numerous enlargeosome-positive cells, the richest and most extensively characterized are those of a PC12 clone, PC12-27, defective of classical neurosecretion. By using ultrastructural immunoperoxidase labeling of formaldehyde-fixed, Triton-X-100-permeabilized PC12-27 cells, we have now identified the enlargeosomes as small vesicles scattered in the proximity of, but never docked to, the plasma membrane. Upon stimulation, these vesicles undergo exocytosis [rapid after the Ca(2+) ionophore, ionomycin, much slower after either the phorbol ester, phorbol myristate acetate (PMA), or ATP, working through a P2Y receptor], with appearance in the plasma membrane of typical desmoyokin/Ahnak (d/A)-positive, Omega-shaped and open profiles evolving into flat patches. Postexocytic removal of the exocytized d/A-positive membrane occurs by two processes: generation of endocytic vesicles, predominant after ionomycin and ATP 100-500 microM; and shedding of membrane-bound cytoplasmic bodies, predominant after PMA and 1 mM ATP, containing little or no trace of endoplasmic reticulum, Golgi, endo/lysosomes and also of a plasma membrane marker. Depending on the stimulation, therefore, the cell-surface expansion by enlargeosome exocytosis is not always recycled but can induce release of specific membranes, possibly important in the pericellular environment.