The subcellular distribution of the polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase (SSAT) was studied in L56Br-C1 cells treated with 10 microM N(1),N(11)-diethylnorspermine (DENSPM) for 24 h. Cells were fractioned into three subcellular fractions. A particulate fraction containing the mitochondria was denoted as the mitochondrial fraction. After DENSPM treatment, an increase in SSAT activity was mainly found in the mitochondrial fraction. Western blot analysis showed an increased level of the SSAT protein in the mitochondrial fraction compared to the cytosolic fraction. Immunofluorescence microscopy and immunogold labeling transmission electron microscopy also showed a mitochondrial association of SSAT. Transmission electron microscopy revealed that the endoplasmic reticulum was devoid of ribosomes in DENSPM-treated cells, in contrast to control cells that contained ample ribosomes. An increased SSAT activity in connection with the mitochondria may be part of the mechanism of DENSPM-induced apoptosis.