NAD-linked ${alpha}$-glycerophosphate dehydrogenase(EC 1.1.1.8) in rat kidney existed as four different isozymic forms with an isoelectric point at pH 6.9, 6.6, 6.4 and 6.2, respectively. The major isozymic form(PI 6.6) was purified using several chromatographic procedures involving an ion exchange and two affinity chromatography steps. The 1,078 fold purified enzyme was homogeneous, nucleotide free protein. The molecular weight of the major isozyme was 62,000 daltons, the optimal pH and temperature for its catalytic activity were 10.0 and $55^{circ}C$, respectively. At the optimal temperature this enzyme was rapidly inactivated$(t_{1/2}=1.5min)$. The major isozyme was activated by the addition of cations such as $K^+$, $Na^+$, $Mg^{++}$ or $Ca^{++}$, and was strongly inhibited by alkylating agents such as P-hydroxymercurybenzoate or iodoacetamide. Glycerophosphate oxidation reaction obeyed the Michaelis-Menton behavior with Km values of 3.22 and 0.31 mM for glycerophosphate and NAD, respectively. The data suggest that although the isozyme pattern of NAD-linked ${alpha}$-glycerophosphate dehydrogenase is quite different by tissue or species of the animal the physico-chemical properties of the major isozymic form are very similar each other except the affinity for substrate and coenzyme.