(2R,3R)-erythro-Fluoromalate, but not the three isomer, is a slow substrate for chicken liver malic enzyme with either NADP or 3-acetylpyridine-NADP (APADP) as the other substrate. The K-m for erythro-fluoromalate is similar to that of malate, but the turnover number with NADP is 3300-fold slower, although 5.5-fold faster with APADP than with NADP. Deuteration of fluoromalate nt C-2 gave an isotope effect on V/K of 1.39 with NADP and 3.32 with APADP. With NADP, the C-13 isotope effects at C-3 were 1.0490 with unlabeled and 1.0364 with deuterated fluoromalate. With APADP, the corresponding values were 1.0138 and 1.0087. These data show that the mechanism is stepwise with both nucleotide substrates, in contrast to the reaction of malate and APADP, which was postulated to be concerted by Karsten et al. [Karsten, W. E., and Cook, P. F. (1994) Biochemistry 33, 2096-2103], a conclusion recently shown to be comet by Edens et al. [Edens, W. A., Urbauer, J. L., and Cleland, W. W. (1997) Biochemistry 36, 1141-1147]. To explain the effect of deuteration on the C-13 isotope effect with APADP, it is necessary to assume a secondary C-13 isotope effect at C-4 on the hydride transfer step of similar to 1.0064 (assuming 5.7 as the intrinsic primary deuterium isotope effect and 1.054 as the product of the C-13 equilibrium isotope effect on hydride transfer and the intrinsic C-13 isotope effect on decarboxylation). The secondary C-13 isotope effect on hydride transfer is thought to result from hyperconjugation between the carbonyl group and C-4 of the enzyme-bound fluorooxaloacetate intermediate.