SUMMARYIn addition to common phenotypes 1, 1–2 and 2 of human deoxyribonuclease I (DNase I), phenotypes 1–3 and 2–3, encoded by a third allele DNASEI*3, have been found by means of isoelectric focusing. The main objective of this study was to identify the mutation site(s) underlying phenotype 3. All eight exons covering the entire open reading frame of the human DNase I structural gene were amplified by the polymerase chain reaction (PCR) and subjected to direct DNA sequencing. When the entire 780‐bp coding region and exon/intron junctions of the DNase I gene of two individuals with phenotypes 1–3 and 2–3 were sequenced, only one nucleotide substitution, a C‐G transition (CCC→GCC), in the codon for amino acid 132 of the mature enzyme located in exon VI was found that resulted in the replacement of proline with alanine (P132A). The mutation was confirmed by allele‐specific amplification of genomic DNA. The replacement of the amino acid residue may reduce the hydrophobicity of the enzyme and thus increase the pi value of the type‐3 isozyme compared with that of type 1, as increasing the hydrophobicity of a protein is known to decrease its pi value. The specific PCR‐amplifications of exons and alleles developed in this study may provide a new tool suitable for rapid screening of DNase I variants.