Roots of the higher plants can assimilate inorganic nitrogen by an enzymatic reduction of the most oxidized form (+6) nitrate to the reduced form (-2) glutamate. For such reactions, the substrates (originated from photosynthates) must be imported to supply energy through the reductant-generating systems within the root cells. Intensive studies over last 70 years (reviewed here) revealed the precise mechanisms of nitrate-to-glutamate transformation in roots with elaborate searches of N-15-tracing, enzymes involved, the reductant-supplying system, and nitrate signaling. In the 1970s, the tracing of N-15-labeled nitrate and ammonia in the roots demonstrated the sequential reduction and assimilation of nitrate to nitrite, ammonia, glutamine amide, and then glutamate. These reactions involve nitrate reductase (NADH-NR, EC 1.7.1.1) in the cytosol, nitrite reductase (ferredoxin [Fd]-NiR, EC 1.7.7.1), glutamine synthetase (GS2, EC 6.3.1.2), and glutamate synthase (Fd-GOGAT, EC 1.4.7.1) in the plastids. NADH for NR is generated by glycolysis in the cytosol, and NADPH for Fd-NIR and Fd-GOGAT are produced by the oxidative pentose phosphate pathway (OPPP). Electrons from NADPH are conveyed to reduce NIR and Fd-GOGAT through Fd-NADP(+) reductase (FNR, EC 1.6.7.1) specifically in the roots. Physiological and molecular analyses showed the parallel inductions of NR, NIR, GS2, Fd-GOGAT, OPPP enzymes, FNR, and Fd in response to a short-term nitrate supply. Recent studies proposed a molecular mechanism of nitrate-induction of these genes and proteins. Roots can also assimilate the reduced form of inorganic ammonia by the combination of cytosolic GS1 and plastidic NADH-GOGAT.