In order to elucidate the causal relationship between increased intracellular free calcium ([Ca2+]i) and induction of apoptosis, serum-free cultured granulosa cell (GC) explants were subjected to various experimental protocols interfering with normal Ca2+ homeostasis. Modulation of apoptotic indices was calculated on DAPI-stained GC explants. In some experiments [Ca2+]i was measured with the Ca2+ probe fura-2 in combination with epifluorescence microscopy. Buffering of [Ca2+]i with BAPTA-AM resulted in inhibition of apoptosis, while increasing extracellular Ca2+ (otherwise called [Ca2+]e load) resulted in a biphasic response characterized by an initial inhibitory effect on apoptosis followed by a delayed phase of increased apoptosis that became apparent at 4 h after withdrawal of the [Ca2+]e load. The initial inhibitory effect of the [Ca2+]e load on apoptosis was dependent on the concentration of the load (range 2-50 mM), was augmented when the [Ca2+]e load was applied in the presence of the Ca2+ channel blocker methoxyverapamil, and was mimicked by applying Mg2+ and Gd3+, two Ca(2+)-receptor agonists. These observations point towards the involvement of an extracellular Ca(2+)-sensing receptor (CaR). Measurements of [Ca2+]i showed that the ion was increased just after [Ca2+]e load, followed by recovery that was complete at 2 h after the load. Collectively these data suggest that a [Ca2+]e load initiates apoptosis, becoming manifest 4 h later, by the provoked [Ca2+]i increase, and this effect is preceded by an apoptosis-inhibiting phase presumably involving CaR activation. We conclude that Ca2+ may act as a first (extracellular) messenger promoting cell survival and as a second (intracellular) messenger activating the cell death pathway.