Ornithine transcarbamylase (OTC) deficiency is an X-linked disease with a heterogeneous phenotype, even in affected males. To detect mutations in the OTC gene using genomic DNA, we have developed a method in which all exons and adjacent introns are amplified and sequenced. Although this approach detected mutations in many cases, the relationship between a mutation and the OTC phenotype was not firmly established. Therefore, we investigated the issue by expression analysis of mutant OTC cDNA in cultured cells. Four mutant OTC cDNAs were constructed, based on the reported cases, using our newly developed method. The normal (wild-type) human OTC cDNA was reproducibly expressed at high levels in these Cos 1 cells. Predicted OTC activities of mutant OTC cDNAs ranged from 0% to 8.9% of the normal level together with variable amounts of the enzyme protein. The predicted enzyme activities account for the clinical phenotype of the disease. Our observations confirm that these mutations are responsible for OTC deficiency in these patients.