Deoxyribonuclease I (DNase I, EC 3.1.21.1) is an endonuclease that preferentially attacks double-stranded DNA in a Ca(2+)-dependent manner to produce oligonucleotides with 5'-phospho and 3'-hydroxy termini. This review deals with the biochemical properties and molecular evolution of DNase I. A comparative study of vertebrate DNase I from Chondrichthyes to Homo sapiens has been carried out. The optimal pH, the role of N-glycosylation, actin inhibition, thermal stability, pH stability, and structure stability are discussed. Moreover, a phylogenetic analysis was performed. The levels of DNase I activity in serum have been suggested to be a critical factor in the initiation of human and mouse SLE. Moreover, as shown above, DNase I is utilized in the treatment of patients with cystic fibrosis. Our comparative study of the biochemical properties and molecular analysis of DNase I will be helpful in the use of DNase I for clinical use.