Microglial cells were harvested from the surface of corpus callosum slices acutely isolated from the brain of neonatal (five- to seven-day-old) mice. Transmembrane ionic currents were measured employing a standard whole-cell voltage-clamp technique. The extracellular application of 1 mM ATP triggered the generation of a complex membrane current comprising three components: (i) an initial fast inward current which had a reversal potential at about -20 to -15 mV; (ii) this initial component was followed by a steady-state inward current with reversal potential about -50 to -40 mV; and (iii) a delayed inward current with a reversal potential close to 0 mV. The first two components (fast and steady-state) had an activation threshold at 10 microM ATP, and 100 microM ATP evoked an almost maximal response. In contrast, the third component of ATP-induced inward membrane current could be observed only while 1 mM ATP was applied. The increase in concentration of tetra-anionic form of ATP (ATP4-) by removal of divalent cations from the bath solution substantially lowered the activation threshold for the delayed component of ATP-induced membrane current; conversely, lowering the ATP4- concentration (by replacing Ca2+ with Mg2+) resulted in its disappearance. These results suggest that ATP4- acts as a true agonist for the activation of the delayed ATP-induced membrane current. We conclude that microglial cells express several purinoreceptor subtypes. The activation of these receptors might play a role in intracellular signal transduction in brain microglia.