Histidinemia (MIM235800) is characterized by elevated histidine in body fluids and decreased urocanic acid in blood and skin and results from histidase (histidine ammonia lyase, EC 126.96.36.199) deficiency. It is the most frequent inborn metabolic error in Japan. Although the original description included mental retardation and speech impairment, neonatal screening programs have identified the majority of histidinemic patients with normal intelligence. Molecular characteristics of histidase in histidinemia have not been determined, and cytogenetically visible deletions of 12q22-24.1 in which histidase gene resides have not been identified in histidinemic patients. In order to investigate whether individuals with this disorder have small deletions, additions, or point mutations in the histidase gene, we screened genomic DNA isolated from 50 histidinemic individuals who were discovered by the neonatal screening program. The methods employed included polymerase chain reaction (PCR) amplification of exons 1-21 of the histidase gene, followed by mutation detection enhancement gel electrophoresis and sequencing of the PCR products displaying heteroduplex bands. Four missense mutations (R322P, P259L, R206T, and R208L), two exonic polymorphisms (T141T c.423A-->T and P259P c.777A-->G), and two intronic polymorphisms (IVS6-5T-->C and IVS9+25A-->G) were identified. The frequencies of each polymorphism estimated either by dot blot allele-specific oligonucleotide hybridization, restriction enzyme digestion, or direct sequencing of the PCR products amplified from 50 unrelated normal individuals were 0.28, 0.30, 0.40, and less than 0.01, respectively. Mutation analysis of one family demonstrated that the patient inherited R322P from the mother and P259L from the father. This report describes the first mutations occurring in the coding region of the histidase structural gene in patients with histidinemia.