It is becoming evident that glia, and astrocytes in particular, are intimately involved in neuronal signaling. Astrocytic modulation of signaling in neurons appears to be mediated by the release of neuroactive compounds such as the excitatory amino acid glutamate. Release of these transmitters appears to be driven by two different processes: (1) a volume regulatory response triggered by hypo-osmotic conditions that leads to the release of osmotically active solutes from the cytoplasm into the extracellular space, and (2) intracellular calcium-dependent vesicle-mediated excytotic release. The regulatory volume decrease may be mediated by any of several different pathways that increase membrane permeability, thus allowing osmolytes to travel down their concentration gradient into the extracellular space. Such pathways include anion channels, hemichannels, P2X receptor channels, and transporters or multidrug resistance proteins. The excytotic release process may use calcium triggered synaptic like vesicle fusion or alterations in constitutive vesicle trafficking to the membrane. Determining the contribution of any of these release mechanisms requires agents that can be used to specifically block pathways of interest. Currently, many of the pharmacological compounds being used exhibit a great deal of cross-reactivity between several of these pathways. For example, the popular anion channel inhibitor 5-nitro-2-(3-phenyl-propylamino)benzoic acid (NPPB) is an efficient blocker of both hemichannels and vesicle loading. This demonstrates the need to more fully characterize the activities of the agents currently available and to choose pathway blockers carefully when designing experiments. © 2004 Wiley-Liss, Inc.