Astrocytes in the rat thalamus display spontaneous [Ca(2+)](i) oscillations that are due to intracellular release, but are not dependent on neuronal activity. In this study we have investigated the mechanisms involved in these spontaneous [Ca(2+)](i) oscillations using slices loaded with Fluo-4 AM (5 microM) and confocal microscopy. Bafilomycin A1 incubation had no effect on the number of spontaneous [Ca(2+)](i) oscillations indicating that they were not dependent on vesicular neurotransmitter release. Oscillations were also unaffected by ryanodine. Phospholipase C (PLC) inhibition decreased the number of astrocytes responding to metabotropic glutamate receptor (mGluR) activation but did not reduce the number of spontaneously active astrocytes, indicating that [Ca(2+)](i) increases are not due to membrane-coupled PLC activation. Spontaneous [Ca(2+)](i) increases were abolished by an IP3 receptor antagonist, whilst the protein kinase C (PKC) inhibitor chelerythrine chloride prolonged their duration, indicating a role for PKC and inositol 1,4,5,-triphosphate receptor activation. BayK8644 increased the number of astrocytes exhibiting [Ca(2+)](i) oscillations, and prolonged the responses to mGluR activation, indicating a possible effect on store-operated Ca(2+) entry. Increasing [Ca(2+)](o) increased the number of spontaneously active astrocytes and the number of transients exhibited by each astrocyte. Inhibition of the endoplasmic reticulum Ca(2+) ATPase by cyclopiazonic acid also induced [Ca(2+)](i) transients in astrocytes indicating a role for cytoplasmic Ca(2+) in the induction of spontaneous oscillations. Incubation with 20 microM Fluo-4 reduced the number of astrocytes exhibiting spontaneous increases. This study indicates that Ca(2+) has a role in triggering Ca(2+) release from an inositol 1,4,5,-triphosphate sensitive store in astrocytes during the generation of spontaneous [Ca(2+)](i) oscillations.