Confocal Ca2+ imaging of rat hippocampal slices shows a paradoxical effect of acute reductions of the [Ca2+]o. Upon slice perfusion with low-Ca2+ media, a prompt intracellular Ca2+ rise selectively occurs in neurones. This response is observed only in slices challenged with agonists of group I metabotropic glutamate or M1 muscarinic receptors. In contrast, the intracellular Ca2+ level of non-stimulated neurones is insensitive to reductions of [Ca2+]o. The phenomenon is observed in 20-25 % of cultured cortical neurones. Evidence is provided demonstrating that: (1) this paradoxical response is not due to a non-specific decrease in divalent cation concentration but it is selectively activated by a reduction in [Ca2+]o, being maximal with [Ca2+]o between 0.25 and 0.5 mM; (2) upon maximal stimulation, 70-90 % of CA1-CA3 pyramidal neurones sense a reduction in [Ca2+]o; a weaker response is observed in neurones from the neocortex, whereas neurones from the dentate gyrus and granule cells from the cerebellum fail to respond; (3) conditions that elicit paradoxical Ca2+ responses cause depolarisation and increase the firing rate of hippocampal neurones; (4) paradoxical Ca2+ rises depend, primarily, on Ca2+ influx through L-type voltage-operated Ca2+ channels and to a lesser extent on release from intracellular Ca2+ stores. Inhibition of phospholipase C or protein kinase C failed to suppress the neuronal response, whereas a selective inhibitor of the Src-family of tyrosine kinases abolishes the paradoxical neuronal Ca2+ rise. A model is presented to explain how this response is elicited by contemporaneous reduction of the [Ca2+]o and metabotropic receptor stimulation; implications for the pathophysiology of the CNS are also discussed.