Astrocytes are increasingly viewed as crucial cells supporting and integrating brain functions. It is thought that the release of gliotransmitters into the extracellular space by regulated exocytosis supports a significant part of communication between astrocytes and neurons. Prior to exocytosis, the membrane-bound vesicles are transported through the astrocyte cytoplasm. Our recent studies have revealed new insights into vesicle trafficking in the cytoplasm of astrocytes and are reviewed in this article. The prefusion mobility of fluorescently labeled peptidergic vesicles was studied in cultured rat and mouse astrocytes. Vesicle delivery to the plasma membrane involved an interaction with the cytoskeleton, in particular with microtubules and actin filaments. Interestingly, vesicle mobility in mouse astrocytes deficient in intermediate filaments show impaired directionality of peptidergic vesicle mobility. To explore whether stimuli that increase the concentration of free calcium ions in the cytoplasm triggered vesicular ATP release from astrocytes, human embryonic kidney-293T cells transfected with a P2X3 receptor were used as sniffers to detect ATP release. Glutamate stimulation of astrocytes was followed by an increase in the incidence of small, transient, inward currents in sniffer cells, reminiscent of postsynaptic quantal events observed at synapses. Some of the membrane-bound vesicles are retrieved from the plasma membrane to be recycled back into the cytosol. Trafficking velocity of postfusion (recycling) atrial natriuretic peptide vesicles was one order of magnitude slower in comparison to the mobility of prefusion vesicles. However, transport of all vesicle types studied required an intact cytoskeleton.