Heritable deficiency of the enzyme argininosuccinate synthetase results in the autosomal recessive disease citrullinemia. RNA was isolated from cultured fibroblasts from citrullinemic patients for synthesis of complementary DNA (cDNA). The mutant mRNAs were reverse transcribed, amplified and cloned for sequencing. The cDNA sequence from one mutant allele revealed the absence of exon 5 sequences, while the sequence from a second mutant allele revealed the absence of exon 6. Genomic DNA from 25 citrullinemic patients was analyzed using intron probes derived from the argininosuccinate synthetase gene. One polymorphic probe located between exons 5 and 6 identified two novel alleles amongst 48 citrullinemia chromosomes examined. Each of these alleles appears to involve deletion of genomic DNA from this region. These two mutations, which were detected as alterations of the gene structure, are the same two mutations that result in deletion of exon 5 or 6, respectively, from the mRNA. These two mutations account for approximately 10% of the chromosomes carrying mutations that cause citrullinemia.