1. Nucleotide-induced currents in untreated (proliferating) and lipopolysaccharide (LPS; 100 ng ml(-1)) treated (non-proliferating) rat microglial cells were recorded by the whole-cell patch-clamp technique. Most experiments were carried out on non-proliferating microglial cells. ATP (100 nM-1 mM), ADP (10 nM-10 mM) and UTP (1 microM-100 mM), but not uridine (100 microM-10 mM) produced a slow outward current at a holding potential of 0 mV. The effect of UTP (1 mM) did not depend on the presence of extracellular Mg2+ (1 mM). The outward current response to UTP (1 mM) was similar in non-proliferating and proliferating microglia. 2. In non-proliferating microglial cells, the ATP (10 microM)-induced outward current was antagonized by suramin (300 microM) or reactive blue 2 (50 microM), whereas 8-(p-sulphophenyl)-theophylline (8-SPT; 100 microM) was inactive. By contrast, the current induced by UTP (1 mM) was increased by suramin (300 microM) and was not altered by reactive blue 2 (50 microM) or 8-SPT (100 microM). 3. The current response to UTP (1 mM) disappeared when K+ was replaced in the pipette solution by an equimolar concentration of Cs+ (150 mM). However, the effect of UTP (1 mM) did not change when most Cl- was replaced with an equimolar concentration of gluconate (145 mM). The application of 4-aminopyridine (1 mM) or Cs+ (1 mM) to the bath solution failed to alter the UTP (1 mM)-induced current. UTP (1 mM) had almost no effect in a nominally Ca2+-free bath medium, or in the presence of charybdotoxin (0.1 microM); the inclusion of U-73122 (5 microM) or heparin (5 mg ml(-1)) into the pipette solution also blocked the responses to UTP (1 mM). By contrast, the effect of ATP (10 microM) persisted under these conditions. 4. I-V relations were determined by delivering fast voltage ramps before and during the application of UTP (1 mM). In the presence of extracellular Cs+ (1 mM) and 4-aminopyridine (1 mM) the UTP-evoked current crossed the zero current level near -75 mV. Omission of Ca2+ from the Cs+ (1 mM)- and 4-aminopyridine (1 mM)-containing bath medium or replacement of K+ by Cs+ (150 mM) in the pipette solution abolished the UTP current. 5. Replacement of GTP (200 microM) by GDP-beta-S (200 microM) in the pipette solution abolished the current evoked by UTP (1 mM). 6. When the pipette solution contained Cs+ (150 mM) instead of K+ and in addition inositol 1,4,5,-trisphosphate (InsP3; 10 microM), an inward current absolutely dependent on extracellular Ca2+ was activated after the establishment of whole-cell recording conditions. This current had a typical delay, a rather slow time course and did not reverse its amplitude up to 100 mV, as measured by fast voltage ramps. 7. A rise of the internal free Ca2+ concentration from 0.01 to 0.5 microM on excised inside-out membrane patches produced single channel activity with a reversal potential of 0 mV in a symmetrical K+ solution. The reversal potential was shifted to negative values, when the extracellular K+ concentration was decreased from 144 to 32 mM. By contrast, a decrease of the extracellular Cl- concentration from 164 to 38 mM did not change the reversal potential. 8. Purine and pyrimidine nucleotides act at separate receptors in rat microglial cells. Pyrimidinoceptors activate via a G protein the enzyme phospholipase C with the subsequent release of InsP3. The depletion of the intracellular Ca2+ pool appears to initiate a capacitative entry of Ca+ from the extracellular space. This Ca2+ then activates a Ca2+-dependent K+ current.