In this study, we describe a novel 420 kDa protein, called Piccolo, found at a wide variety of adult rat brain synapses. High protein levels in the cerebellum, the olfactory bulb and the hippocampus were frequently observed to be associated with asymmetric type 1 synapses. Piccolo is selectively enriched in presynaptic terminals, but is not a component of synaptic vesicles (SVs). Immunogold electron microscopy revealed that Piccolo localizes to the amorphous material among SVs at the presynaptic plasma membrane. Biochemical studies showed that it is very tightly bound to this structure. Thus, we speculate that Piccolo is a structural component of the presynaptic cytomatrix which anchors SVs to the presynaptic plasmalemma.