Locations of a distinctive mode of trans-endocytosis involving dendrites, axons, and glia were quantified through serial section electron microscopy. Short vesicular or long vermiform evaginations emerged from dendrites and axons and were engulfed by presynaptic or neighboring axons, astrocytes, and, surprisingly, a growth cone to form double-membrane structures called spinules. In total, 254 spinules were evaluated in 326 microm(3) of stratum radiatum in area CA1 of mature rat hippocampus. Spinules emerged from spine heads (62%), necks (24%), axons (13%), dendritic shafts (1%), or nonsynaptic protrusions (<1%) and invaginated into axons (approximately 90%), astrocytic processes (approximately 8%), or a growth cone (approximately 1%). Coated pits occurred on the engulfing membrane at the tips of most spinules (69%), and double-membrane structures occurred freely in axonal and astrocytic cytoplasm, suggesting trans-endocytosis. Spinule locations differed among mushroom and thin spines. For mushroom spines, most (84%) of the spinules were engulfed by presynaptic axons, 16% by neighboring axons, and none by astrocytic processes. At thin spines, only 17% of the spinules were engulfed by presynaptic axons, whereas 67% were engulfed by neighboring axons and 14% by astrocytic processes. Spinules engulfed by astrocytic processes support the growing evidence that perisynaptic glia interact directly with synapses at least on thin spines. Spinules with neighboring axons may provide a mechanism for synaptic competition in the mature brain. Trans-endocytosis of spinules by presynaptic axons suggest retrograde signaling or coordinated remodeling of presynaptic and postsynaptic membranes to remove transient perforations and assemble the postsynaptic density of large synapses on mushroom spines.