The amino acid replacements in five missense mutant forms of Neurospora crassa NADP-specific glutamate dehydrogenase, am 1, am 2, am 3, am 7 and am 19, have been identified. All these mutations cause an effective loss of enzyme activity in vivo, either by disruption of a pH-dependent conformational equilibrium between inactive and active states ( am 2, am 3 and am 19) or by abolition of coenzyme binding ( am 1 and am 7). The altered residues have been identified in tryptic or chymotryptic peptides fractionated from digests of the intact polypeptide chain, as Lys141 → Met ( am 19), His142 → Gln ( am 2), Ser336 → Phe ( am 1), Gly372 → Ser ( am 7) and Glu393 → Gly ( am 3). In addition, one prototrophic revertant from am 2 and four from am 19 all appear to be due to partially compensating replacements at second residues, two of which have been identified in revertants of am 19 as Ile191 → Leu ( am 19R1) and Gln191 → Arg ( am 19R24). The apparently complete defect in NADPH binding caused by the am 1 substitution supports the identification of a coenzyme-binding domain previously postulated by Wootton (1974). A plausible stereochemical explanation of the effect of this substitution is proposed, based on considerations of homology with glyceraldehyde 3-phosphate dehydrogenase. It is proposed that the conditional defect in coenzyme binding caused by the am 7 substitution, which unlike the am 1 substitution lies outside the proposed coenzyme binding domain, is an indirect conformational effect. The conformationally defective mutants am 2, am 19 and am 3 show drastic destabilization of the active allosteric conformation, indicating that at least two sites in the molecule, around residues 141 to 142 and 391 to 393, are important for the maintenance of normal allosteric equilibrium. The second residue replacement in am 19R24 suggests that an interaction, probably involving charged residues, occurs between these two regions of the polypeptide chain. The positions of the amino acid replacements in the primary mutants are colinear with those of the corresponding sites in the genetic map, which was constructed using the unconventional criterion of conversion polarity. Recombination frequencies are roughly proportional to physical distance but with some marker effects and an indication of map expansion.