Bcl-x, a potent regulator of cellular decisions of life and death, has multiple survival-enhancing activities that rely on distinct protein regions. Evidence suggests that depending on the local environment and the binding of protein or peptide partners, Bcl-x can take on several conformations that expose different protein regions. However, biological occurrence of conformational forms has been very difficult to study, because structure determination techniques use large quantities of protein, purified under conditions that change Bcl-x conformation. We show here that standard 2D isoelectric focusing techniques can be used to distinguish conformationally distinct forms of Bcl-x in cell lysates. Conformational isoelectric forms were manipulated through the use of detergents and buffers of differing pH. Our data indicate that post-translational modifications are not needed for or associated with conformational changes, distinguishing the dominant isoelectric forms of Bcl-x. We found that Bcl-x conformational isoelectric forms have preferred subcellular localization patterns. Moreover, conformational forms are differently regulated in certain locations during cytokine starvation of IL-3-dependent cells. Therefore, we provide evidence that 2DIEF can be used to view biologically distinct conformational differences in Bcl-x on minute quantities of unpurified protein from cells or lysates.