Ornithine transcarbamylase (OTC) is an X-linked, liver-specific enzyme that catalyzes the second step of the urea cycle. In humans, inherited deficiency of OTC in hemizygous affected males usually results in severe ammonia intoxication and early death. To characterize mutations responsible for OTC deficiency, we used the PCR to amplify cDNAs prepared from patient livers which demonstrated no OTC enzyme activity and no OTC cross-reacting material on western blots. In three of seven cases, smaller than normal products were observed. Sequencing of these cDNAs revealed that two were missing exon 7 of the OTC gene and that the other was missing the first 12 bp of exon 5. Sequencing of genomic DNA from these three patients revealed that one mutant missing exon 7 had a T-to-C substitution in the 5' splice donor site of intron 7. The other mutant missing exon 7 had an A-to-G change in the third position of intron 7. It is interesting that both of these mutations resulted in skipping the preceding exon rather than in inclusion of some or all of the affected intron. In the third mutant, an A-to-T substitution was found in the 3' splice acceptor site at the end of intron 4. Here, a cryptic splice acceptor site within exon 5 was used. Northern blotting of liver RNA from these patients demonstrated (a) reduced, but significant, amounts of OTC mRNA in one of the patients who had a deleted exon 7 but (b) very little OTC mRNA in the other two patients. We propose that these point mutations, which result in aberrant splicing of the OTC pre-mRNAs, lead to OTC deficiency through either decreased efficiency of mRNA export from the nucleus to the cytosol or synthesis of enzyme subunits that are unstable and rapidly degraded. We speculate that abnormal mRNA splicing may represent a relatively common mechanism in the pathogenesis of this disease.