Active-site mutants of glutamate dehydrogenase from Clostridium symbiosum have been designed and constructed and the effects on coenzyme preference evaluated by detailed kinetic measurements. The triple mutant F238S/P262S/D263K shows complete reversal in coenzyme selectivity from NAD(H) to NADP(H) with retention of high levels of catalytic activity for the new coenzyme. For oxidized coenzymes, kcat/Km ratios of the wild-type and triple mutant enzyme indicate a shift in preference of approximately 1.6 × 107-fold, from ∼ 80 000-fold in favour of NAD+ to ∼ 200-fold in favour of NADP+. For reduced coenzymes the corresponding figure is 1.7 × 104-fold, from ∼ 1000-fold in favour of NADH to ∼ 17-fold in favour of NADPH. A fourth mutation (N290G), previously identified as having a potential bearing on coenzyme specificity, did not engender any further shift in preference when incorporated into the triple mutant, despite having a significant effect when expressed as a single mutant.