ATP has been proposed as a possible chemical mediator of synaptic transmission in the spinal dorsal horn on the basis that it is released in dorsal horn synaptosomes in a Ca(2+)-dependent manner and that its effects mimic those of synaptic inputs to dorsal horn neurons. In the present study we examined the actions of ATP on neurons and glia in cell culture using optical and electrophysiological recording techniques. We found that ATP increased intracellular Ca2+ concentration ([Ca2+]i) in > 99% of astrocytes. In contrast, only 35% of neurons and 20% of oligodendrocytes responded to ATP. The prevalence of the ATP-evoked response in astrocytes led us to characterize the type of receptor mediating the response, the source of Ca2+, and the membrane currents activated by ATP. We found that ADP was approximately equipotent with ATP in increasing [Ca2+]i whereas AMP and adenosine had no effect. In addition, responses to ATP were blocked in a concentration-dependent manner by the P2 purinergic receptor antagonist suramin. Furthermore, as it was found that 2-methylthio-ATP was more potent than ATP and that beta, gamma-methylene-ATP was ineffective, the responses were mediated via the P2 gamma subtype of purinergic receptor. The increase in [Ca2+]i evoked by ATP persisted in extracellular medium with no added Ca2+ and containing EGTA, indicating that this increase was due to release of Ca2+ from intracellular stores. Release of Ca2+ by ATP was blocked by thapsigargin but was unaffected by caffeine. ATP had several effects on membrane current activating inward, outward, and mixed currents despite uniformly causing increases in [Ca2+]i. These observations indicate that ATP has diverse electrophysiological effects on astrocytes as well as increasing [Ca2+]i in these cells. We speculate that ATP released from synaptic terminals in the dorsal horn might act not only on postsynaptic neurons but also on perisynaptic astrocytes. Thus, a physiological role for ATP may be as a neuronal-glial signaling molecule within the spinal dorsal horn.