The mode of action of fenpyroximate in Tetranychus urticae Koch (two-spotted spider mite) was studied biochemically and morphologically. Biochemical assays showed that an adult female T. urticae contained 88.96 ± 10.32 pmol ATP, which rapidly decreased to 74.8, 31.2, and 2.9%, 60 min after spraying with 0.05, 0.5, and 5 μg/ml fenpyroximate, respectively. The mortalities at that time were 2, 18, and 46%, respectively. In vitro experiments using rat liver mitochondria showed that 10 μMfenpyroximate inhibited electron transport when NADH or NADH-linked substrates were used as the electron donors but not when succinate was used as the substrate. Therefore, the site of inhibition on the electron transport chain was considered to be the NADH-coenzyme Q (Co Q) reductase. Mitochondrial NADH-Co Q reductase of T. urticae was also inhibited by fenpyroximate, and the I50 value was estimated to be 0.08 μM. Transmission electron microscopy observations of T. urticae sprayed with 0.5 μg/ml fenpyroximate showed that the compound caused morphological changes in mitochondria in peripheral nerve cells such as swelling, irregular cristae arrangement, and lower matrix electron density. Similar morphological changes in mitochondria were also obvious in the ovaries and epidermal cells, but not in muscular cells or central nervous mass cells. These results indicate that the inhibition of mitochondrial NADH-Co Q reductase by fenpyroximate seems to induce a decrease in ATP contents and morphological changes in mitochondria. Ultimately, this would contribute to the acaricidal and knockdown activities against T. urticae by this compound.