Since the cloning of the cDNA for X-linked ornithine transcarbamylase (OTC) in 1984, diagnostic accuracy of OTC deficiency for prenatal and carrier detection has been greatly improved by the use of linkage analysis. However, the use of RFLP-based diagnosis is limited in this and in other new mutation diseases. Here we report both the use of direct mutation detection by new PCR-based techniques and our experience with linkage-based diagnosis in 18 families. We have previously reported the use of chemical mismatch cleavage to detect mutations first in amplified mRNA and then in genomic DNA of patients. This technique has now been utilized for prenatal diagnosis. Primers for specific amplification of OTC exons 1, 3, 5, 9, and 10 have been developed and been employed to map deletions of the OTC gene in two families. These primers also have been used to detect alterations in the TaqI sites found in exons 1, 3, 5, and 9. Four novel mutations of the OTC gene leading to abolition of a TaqI site in the OTC cDNA were discovered. One of these mutations is in exon 1; two lie in exon 3; and one is in exon 9. In addition, we have used the PCR products as probes to identify the exon-specific bands seen on Southern blots and to map the polymorphic BamHI and MspI sites, which are commonly used for linkage analysis. This information will facilitate the interpretation of altered band patterns seen in deletion cases and in cases of point mutations affecting restriction sites. Utilization of the appropriate combination of these molecular techniques permitted accurate diagnostic evaluations in 17 of 18 families.