Glycogen synthase kinase-3 beta (GSK3 beta) is located predominantly in the cytosol, but also is in nuclei and mitochondria. In SH-SY5Y cells, primary cortical neurons, and mouse brain, the portion of active GSK3 beta (not phosphorylated on serine-9) was 5- to 8-fold greater in nuclei and mitochondria than in cytosol. Correspondingly greater GSK3 beta activities were measured in nuclei and mitochondria compared with cytosol. Stimulation of apoptotic signaling by treatment with camptothecin or thapsigargin activated GSK3 beta in the nucleus and mitochondria, but not the cytosol, whereas inhibition of GSK3 beta by lithium treatment affected all three pools of GSK3 beta. Thus, the nuclei and mitochondria contain disproportionately high levels of active GSK3 beta, which is selectively further activated by some apoptotic stimuli.