The mechanisms of neuronal degeneration following hypoxia/ischemia remain undefined, but the processes include increases in neurotransmitter release, elevation of cytosolic-free calcium concentration, and changes in signal transduction pathways. Activation of the multigene family of protein kinase C (PKC) has been associated with the release of neurotransmitter and the survival of neurons. Therefore, to understand which PKC isozymes are involved in hypoxia/ischemiainduced neuronal degeneration, we examined PKC isozymes after chemical hypoxia (i.e., KCN exposure) in PC12 cells. Cell toxicity, as measured by lactate dehydrogenase (LDH) release, was increased significantly by KCN in glucose-free DMEM and was exaggerated by acute 12-O-tetradecanoyl phorbol-13acetate (TPA) pretreatment. Under parallel conditions, KCN elevated cytosolic-free calcium ([Ca 2ุ ] i ) in glucose-free but not in glucose containing DMEM, and TPA pretreatment did not exaggerate KCN's effect on [Ca 2ุ ] i . Thus, increases in [Ca 2ุ ] i are not sufficient for the synergistic toxic effect of KCN and TPA. In the glucose-free DMEM, selective PKC isozyme inhibitor Go 6976 at 10 nM completely inhibited KCN-induced LDH release and at higher concentrations (1 M) inhibited the basal levels of LDH release. The protein levels of PKCs in the nuclear, membrane, and cytosolic fractions were measured by Western blot analysis using antibodies against specific isoforms. Two Ca 2ุ -dependent (ุโฃ, ุโฅ) and four Ca 2ุ -independent (ุโฆ, ุโ, ุ, and ุ) isozymes were identified and two isozymes (ุโค and ุ) were not detected in the subcellular fractions of PC12 cells. Treatment of the cells with TPA significantly activated translocation of conventional PKC-โฅ from the cytosol to the membrane and nuclear fractions and other PKC isozymes (ุโฃ, ุโฆ, and ุโ) from the cytosol to the membrane, but not atypical PKC-and -. Although only the levels in the nuclear PKC-โฅ but not other PKC isozymes were increased significantly following KCN, the levels of cPKC-โฃ and -โฅ in the membrane mainlyand those and PKC-โ in the nucleus-were increased when KCN was combined with TPA. In addition, this condition (TPA ุ KCN) did not affect the TPA insen-sitive atypical isozymes, PKC-or -. Taking the results together, differential activation/translocation of PKC isozymes by KCN and TPA is important in the regulation of chemical hypoxia-induced cell injury in PC12 cells.