Argininosuccinic aciduria is an urea cycle disorder caused by argininosuccinate lyase (ASL) deficiency and is inherited as an autosomal-recessive trait. To date, mutation analysis has been limited because of incomplete sequence information about the human ASL gene. As a consequence, only 12 different mutations in 12 patients have been reported, so far. This study aimed at the completion of the structure and the sequence of the human ASL gene, the development of a genomic DNA-based system for mutation analysis and, finally, the characterisation of the molecular genetic background of ASL deficiency in 27 unrelated patients. This report provides transcript variants, the complete sequence of the human ASL gene and a complete ASL homologue on chromosome 22. The homologue was formerly thought to be a pseudogene but was found, in this study, to be correlated with an immunoglobulin-lambda-like mRNA. On the basis of the novel sequence data, a polymerase reaction chain system for mutation-screening in all 16 coding exons of the ASL gene was established and applied to the analysis of the ASL-deficient patients. We found mutations in all of the 54 investigated alleles and identified 23 (19 novel) different mutations. Some mutational hot-spots were identified (mainly in exons 4, 5, and 7) as were several predominant mutations: IVS5+1G-->A (15 alleles), c.532G-->A (7), c.346C-->T (6), c.1153C-->T (4). This study introduces a system for mutation analysis in the ASL gene, thereby elucidating the genetic background of ASL deficiency, which was found to be associated with considerable allelic heterogeneity.