Various fatty acids containing 10--22 carbons and including unsaturated derivatives were found to be inhibitors of rabbit liver and skeletal muscle sn-glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8). For the liver enzyme, the logarithm of the inhibition constant was linearly related to the number of carbon atoms in the saturated fatty acids whereas the muscle enzyme, which was generally more strongly inhibited, showed a nonlinear dependence. The liver and muscle enzymes also interacted differently with a series of unsaturated fatty acids for which a high degree of specificity was exhibited which was related to the position, configuration, and number of double bonds in the compound. A steady-state kinetic analysis shows that under some conditions, the kinetics of the NADH reduction of dihydroxyacetone phosphate by NADH in the presence of stearic acid do not follow simple Michaelis-Menten behavior but rather the velocity shows a sigmoidal dependence on fatty acid concentration and strong substrate inhibition. Stearic acid is a much poorer inhibitor of the NAD-dependent oxidation of glycerol-3-phosphate. At low substrate concentrations stearic acid is competitive with respect to NAD with an inhibition constant of 24 micrometer for stearic acid. In addition to the effect of fatty acids on the initial velocities of the enzyme-catalyzed reactions, preincubation of the enzyme with fatty acid leads to a slow, time-dependent irreversible inactivation of the enzyme which is prevented by the presence of NADH. The results are discussed in terms of the differences in the conformations of the hydrophobic binding sites on the two enzymes.