Protein kinase C (PKC) isozymes constitute a family of at least 12 structurally related serine-threonine kinases that are differentially regulated and localized, and are presumed to mediate distinct intracellular functions. To explore their roles in intact cells, investigators are developing cell-permeable, isoform-selective inhibitors. 2,2',3,3',4,4'-Hexahydroxy-1, 1'-biphenyl-6,6'-dimethanol dimethyl ether (HBDDE) is reported to be a selective inhibitor of PKC alpha and gamma with IC(50) values of 43 and 50 microM, respectively, using an in vitro assay. However, data examining the potency and selectivity of HBDDE in intact cells are lacking. Employing rodent cerebellar granule neurons as a model system, we investigated the effects of HBDDE using cell survival as a functional end-point. HBDDE induced an apoptotic form of cell death that was dependent upon protein synthesis and included activation of a terminal executioner of apoptosis, caspase 3. The concentration of HBDDE required for half-maximal cell death was less than 10 microM ( approximately 5-fold less than the reported IC(50) values for PKC alpha and gamma in vitro). Furthermore, HBDDE induced apoptosis even after phorbol-ester-mediated down-regulation of PKC alpha and gamma, indicating that this effect is independent of these isoforms. Consistent with this, 2-[1-(3-dimethylaminopropyl) indol-3-yl]-3-(indol-3-yl)-maleimide (GF 109203X), a general inhibitor of all classical and some novel PKCs, did not interfere with survival. Thus, HBDDE should not be used as an isoform-selective inhibitor of PKC alpha or gamma in intact cells. Nevertheless, identification of its target in granule neurons will provide valuable information about survival pathways.