Maternal smoking during pregnancy is often associated with a decrease in placental function, which might lead to intrauterine growth retardation. Because tobacco is known to alter the mitochondrial respiratory function in cardiomyocytes and lung tissue, we hypothesized that placental mitochondrial function could be altered by maternal smoking. Placental mitochondria from 9 smoking and 19 nonsmoking mothers were isolated by differential centrifugation. Mitochondrial oxygen consumption was measured by polarography, and the enzymatic activity of each complex of the electron transport chain was assessed by spectrophotometry. In addition, the relative content in mitochondrial DNA (mtDNA) was determined by real-time quantitative PCR in placentas from seven smoking and seven nonsmoking mothers. We observed a 29% reduction in the enzymatic activity of complex III in the placental mitochondria from smokers compared with nonsmokers (P = 0.03). The relative content of mtDNA (with respect to the beta-globin gene) was reduced by 37% in the placental tissue from smokers compared with nonsmokers (P < 0.02). Both the enzymatic activity of complex III and mtDNA content were inversely related with the daily consumption of cigarettes, and mtDNA content was correlated with cord blood insulin-like growth factor-binding protein-3 (r = 0.74, P < 0.01), a marker of fetal growth. These results show that maternal smoking is associated with placental mitochondrial dysfunction, which might contribute to restricted fetal growth by limiting energy availability in cells.