The ability of the Bcl-2 family of anti-apoptotic proteins to prevent programmed cell death has been attributed to multiple mechanisms, including binding pro-apoptotic proteins as well as the formation of pores. The evidence for the latter mechanism is extensive but indirect and includes the presence of alpha helical domains (designated alpha-5 and alpha6) that are similar to several bacterial pore forming domains, as determined by NMR and Xray crystallography of recombinant Bel-XL without the hydrophobic insertion tail; the ability of recombinant Bel-XL and Bcl-2 to form ion conducting channels in artificial lipid bi-layers; and the loss of anti-apoptotic activity of a Bel-XL mutant in which a portion of the putative pore forming domain is altered. To assess the conformation of Bcl-2, we have examined the local environment of cysteine 158 located in the alpha-5 helix near the base of the pore forming region, and cysteine 229 located in the hydrophobic insertion sequence, using the lipid impermeant cysteine modifying agent iodoacetylamino stilbene disulfonic acid (IASD). When human Bcl-2 is translated in vitro in the absence of membranes, both cysteines are modified by IASD, albeit with different kinetics. In the presence of membranes, cysteine 229 is not labelled by IASD, consistent with our previous work demonstrating that the insertion sequence is necessary and sufficient for insertion into multiple intracellular membranes. In this circumstance, cysteine 158 is still modifiable by IASD. In the Rat 1/myc model system of apoptosis, cysteine 158 is accessible to IASD in untreated cells, and early during serum starvation-induced apoptosis at time points during which Bcl-2 is protective. By contrast, late in apoptosis when Bcl-2 has lost its effect, this cysteine is no longer modifiable. Cysteine 229 remains inaccessible under all three conditions, consistent with the expected membrane topology of Bcl-2 in cells. Ongoing experiments will assess whether a functionally active Bel-XL mutant targeted to the endoplasmic reticulum in human breast cancer MCF7 cells, and endogenous Bcl-2 in the lymphocytes of patients with B cell chronic lymphocytic leukemia adopt a similar configuration. Our results suggests that the alpha-5 helical loop is not in a lipid environment while Bcl-2 is functioning to prevent apoptosis.