Cultured astrocytes can release a variety of messenger substances via receptor-mediated mechanisms, implicating their potential for regulated exocytosis and the participation of proteins of the SNARE complex. Here we demonstrate the astrocytic expression and organellar association of a large variety of synaptic proteins (synaptobrevin II, synaptotagmin I, synaptophysin, rab3a, synapsin I, SNAP-25, and syntaxin I) and also of the ubiquitous cellubrevin. As revealed by immunoblotting the expression of synaptic proteins was highest within the first few days after plating. Synaptophysin and SNAP-25 showed the most significant decline with prolonged culture time. Rab3a and synaptobrevin II were retained at a high level and synaptotagmin I, synapsin I, and syntaxin I at a lower level until 20 DIV. The immunoreaction for cellubrevin was low at the beginning and increased with prolonged culture time. As revealed by light microscopical immunocytochemistry the proteins are expressed by GFAP-positive astrocytes and associated with organelles of varying size. Immunoelectron microscopical analysis allocates synaptobrevin II and synaptophysin to the membranes of vesicular organelles. Double labeling experiments for pairs of synaptic proteins reveal that individual synaptic proteins can be entirely colocalized or partly reside on different organelles. Subcellular fractionation of astrocyte cultures by sucrose density gradient centrifugation after 2, 6, 13, and 20 DIV showed that the proteins sediment with ATP containing organelles of a broad density range. Our data suggest that messenger substances may be released from cultured astrocytes via receptor-mediated, Ca2+-dependent exocytosis.