Glucocorticoids induce apoptosis in a variety of cells. It has been described that glucocorticoids activate calcineurin (PP-2B) which in turn stimulates apoptosis by dephosphorylation of BAD. Previously, we described that the synthetic glucocorticoid dexamethasone (100 nM dexa) induces apoptosis in insulin secreting cells. The aim of the present study was to examine whether activation of PP-2B and dephosphorylation of BAD might be involved in dexa-induced apoptosis. INS-1 cells were cultured under standard conditions. Apoptosis was quantified by DAPI-stained condensed nuclei, TUNEL assay and by caspase-3 activity. Dexa increased apoptosis significantly more at 20 mM (10-fold) than at 5 mM glucose (5-fold). RU486, the glucocorticoid receptor (GR) antagonist, inhibited the dexa effect. Two PP-2B inhibitors, FK506 and deltamethrin, reduced dexa-mediated apoptosis by 34% and 72%, respectively. Dexa did not increase cytosolic Ca2+ activity measured with the Ca2+ indicator fura-2. PP-2B activity, measured in INS-1 cell homogenates, was stimulated by dexa 2-fold more at high glucose than at low glucose. Geldanamycin, an antagonist of HSP (heat shock proteins) inhibited stimulation of PP-2B by 80%. Dexa decreased phosphorylation of BAD. These data suggest that dexa-induced apoptosis in INS-1 cells may involve PP-2B stimulation by HSP90 released from the GR complex and may be enhanced by glucose induced Ca2+ entry.