Glucocorticoids are known to induce apoptosis in lymphoid cells, and Bcl-2 overexpression can block the apoptosis-inducing action of glucocorticoids. Since phosphorylation of Bcl-2 is implicated in regulating Bcl-2 function, we considered the role of Bcl-2 phosphorylation in protecting lymphoid cells from glucocorticoid-induced cell death. Five stably transfected cell lines of WEHI 7.1 cells expressing either wild-type Bcl-2 or alanine mutants of Bcl-2 at amino acids threonine 56, serine 70, threonine 74, or serine 87 were created. Expression of the mutant Bcl-2 proteins was documented by flow cytometry and Western blot analysis. Mutation of Bcl-2 on T56 and S87 eliminated the ability of Bcl-2 to inhibit glucocorticoid-induced cell shrinkage, mitochondrial depolarization, DNA fragmentation, and cell death. Mutation of T74 only partially impaired the ability of Bcl-2 to block glucocorticoid-induced apoptosis whereas mutation of S70 in Bcl-2 did not alter its ability to block glucocorticoid-induced apoptosis.