The mechanism of hydrogen incorporation into fatty acids was investigated with intact Escherichia coli cells, a crude enzyme preparation and purified reductases of fatty acid synthetase system. The distributions of deuterium atoms incorporated into fatty acids from 2H2O and stereospecifically deuterium-labeled NADPH or NADH were determined by mass spectrometry. When E. coli was grown in 2H2O, almost every hydrogen atom of cellular fatty acids was incorporated from the medium. When fatty acids were synthesized from acetyl-CoA, malonyl-CoA and NADPH in the presence of a crude enzyme preparation of either E. coli or Bacillus subtilis, almost every hydrogen atom was also incorporated from the medium. In contrast to these results, purified beta-ketoacyl acyl carrier reductase directly transferred the HB hydrogen of NADPH to beta-ketoacyl acyl carrier protein, and purified enoyl acyl carrier protein reductase also transferred the HB hydrogen of NADPH and NADH directly to enoyl acyl carrier protein. In the crude enzyme preparation of E. coli, we found high activities which exchanged the HB hydrogen of NADPH with the deuterium of 2h2o. the conflicting results of the origin of hydrogen atoms of fatty acids mentioned above are explained by the presence of enzymes, which catalyzed the rapid exchange of NADPH with the deterium of 2H2O prior to the reaction of fatty acid synthetase.