Linkage of certain neurological diseases to Na(+) pump mutations and some mood disorders to altered Na(+) pump function has renewed interest in brain Na(+) pumps. We tested nanomolar ouabain on Ca(2+) signalling (fura-2) in rat hippocampal neurone-astrocyte co-cultures. The neurones and astrocytes express Na(+) pumps with a high-ouabain-affinity catalytic subunit (α3 and α2, respectively); both also express pumps with a ouabain-resistant α1 subunit. Neurones and astrocytes were identified by immunocytochemistry and by stimulation; 3-4 μM L-glutamate (Glu) and 3 μM carbachol (CCh) evoked rapid Ca(2+) transients only in neurones, and small, delayed transients in some astrocytes, whereas 0.5-1 μM ATP evoked Ca(2+) transients only in astrocytes. Both cell types responded to 5-10 μM Glu or ATP. The signals evoked by 3-4 μM Glu in neurones were markedly inhibited by 3-10 μm MPEP (blocks metabotropic glutamate receptor mGluR5) and 10 μm LY341495 (non-selective mGluR blocker), but not by 80 μm AP5 (NMDA receptor blocker) or by selective block of mGluR1 or mGluR2. Pre-incubation (0.5-10 min) with 1-10 nm ouabain (EC50 < 1 nm) augmented Glu- and CCh-evoked signals in neurones. This augmentation was abolished by a blocker of the Na(+)-Ca(2+) exchanger, SEA0400 (300 nm). Ouabain (3 nm) pre-incubation also augmented 10 μM cyclopiazonic acid plus 10 mm caffeine-evoked release of Ca(2+) from the neuronal endoplasmic reticulum (ER). The implication is that nanomolar ouabain inhibits α3 Na(+) pumps, increases (local) intracellular Na(+), and promotes Na(+)-Ca(2+) exchanger-mediated Ca(2+) gain and increased storage in the adjacent ER. Ouabain (3 nm) also increased ER Ca(2+) release and enhanced 0.5 μM ATP-evoked transients in astrocytes; these effects were mediated by α2 Na(+) pumps. Thus, nanomolar ouabain may strongly influence synaptic transmission in the brain as a result of its actions on the high-ouabain-affinity Na(+) pumps in both neurones and astrocytes. The significance of these effects is heightened by the evidence that ouabain is endogenous in mammals.