Properties of mutant δ-aminolevulinate dehydratase (ALAD) found in patients with ALAD porphyria were studied by enzymological and immunological analyses after the synthesis of enzyme complexes using a cell-free system. Enzyme activities of homozygous G133R, K59N/G133R, V153M, and E89K mutants were 11%, 22%, 67%, and 75% of the wild-type ALAD, respectively, whereas that of K59N, a normal variant, was 112%. Enzyme activities of L273R, C132R and F12L were undetectable. Co-synthesis of F12L, L273R, G133R, K59N/G133R, or C132R mutants with the wild-type at various ratios showed that ALAD activity was proportionally decreased in the amount of the wild-type in the complex. In contrast, co-synthesis of V153M, K59N, and E89K with the wild-type did not influence enzyme activity of the wild-type. Surface charge changes in K59N, E89K, C132R and G133R predicted by mutations were also confirmed by native polyacrylamide gel electrophoresis. A compound E89K and C132R complex showed ALAD activity similar to that was found in erythrocytes of the patient. These findings indicate that cell-free synthesis of ALAD proteins reflects enzymatic activities found in patients, and suggest that, in addition to the direct effect of mutations on the catalytic activity, conformational effects play an important role in determining enzyme activity.