Alterations in intracellular free calcium concentration ([Ca2+]i) are instrumental in apoptosis. We have previously shown that a [Ca2+]i increase above 1000 nM is related to the appearance of apoptosis in serum-free cultures of granulosa cell sheets. In the present study examined how the [Ca2+]i increase relates to indicators of distinct phases of the apoptotic cascade. We used a double staining technique whereby loading with the Ca2+ indicator fura-2 and capture of a [Ca2+]i image, was followed by staining with annexin-V, as an early apoptotic marker or with acridine orange, marking the late degradation phase. Calcium imaging showed a large heterogeneity of cellular [Ca2+]i levels. [Ca2+]i was moderately increased to approximately 230 nM in annexin positive cells but was at resting level in cells with nuclear manifestations of apoptosis as evidenced by acridine orange. Our results suggest that a modest [Ca2+]i increase is related to phosphatidylserine translocation and that [Ca2+]i has already recovered in apoptotic cells displaying chromatin condensation and/or nuclear fragmentation. Granulosa cells with [Ca2+]i above 1000 nM were never observed to strain positive for the apoptotic markers used; therefore, large [Ca2+]i increases are probably related to the apoptosis initiation phase occurring upstream of phosphatidylserine exposure.